Combined Radiations: Biological Effects of Mixed Exposures Across the Radiation Spectrum

1. Introduction

Different types of radiation can be combined in various contexts, whether therapeutic or experimental; they may be applied to biological or non-biological targets, simultaneously or sequentially, with intervals between irradiations ranging from minutes to weeks. The dose of each combined radiation type is another important factor, as are other dosimetric features of a given experimental configuration. For biological targets, a number of measurable endpoints are used to assess the induced effects, which are typically the primary focus of related studies. “Combining different radiation types” thus constitutes a broad set of practices, involving a variety of methods developed and applied for distinct purposes.

Although some narrative reviews exist focusing individually on conventional radiobiology, space radiobiology or therapeutic radiation combinations, to our knowledge, none provides a systematic analysis of combined exposure types across biological systems and application domains. In many respects, this systematic review pursued an ambitious aim: to map a curated body of studies on conventional and space radiobiology, as well as the therapeutic use of combined radiation exposures, analyzing their qualitative and quantitative features.

More specifically, this review aimed to identify, categorize, summarize, and analyze the results of eligible studies involving biological systems (human, animal, and in vitro) exposed to mixed radiation types in therapeutic, conventional and space radiobiological contexts. As a result, it provides a concise overview of the diverse processes and outcomes that define the multifaceted applications of combined radiation exposure in biological systems, reflecting the current scientific understanding of the field.

2. Methods

The challenges impeding a rapid compilation of all relevant publications required for such an extensive review arise from a range of factors, including the dispersion of publications across a wide variety of databases, shifting terminology and evolving epistemological frameworks. The selection criteria applied during the literature review are presented here to clarify the rationale behind the inclusion of each study in the compiled data set. First, we determined which types of combined radiations to include—specifically, which wavelength and/or energy ranges were relevant. Our focus aligned with conventional and space radiobiology, as well as several combined therapeutic modalities. Consequently, other forms of mixed exposure, such as environmental exposure to extremely low-frequency electromagnetic fields (ELF-EMF), microwave radiation, or radiofrequency electromagnetic fields (RF-EMF) in combination with other types, were knowingly excluded from this review. Similarly, combinations involving radiation and non-radiation agents, such as chemicals or microgravity, were also excluded, as they fall outside the scope of radiation–radiation interactions. In rare cases where non-radiation factors were present (e.g., the NASA Twins Study), the inclusion of those publications was primarily justified by their focused investigation of combined radiation exposures, with any additional variables considered secondary.

Regarding the combinatorial nature of the exposures, studies involving the sequential application of radiations with time intervals longer than those typically defining genuine combinatorial exposure (e.g., >24 h) were mostly excluded. That said, nearly all included studies involved either simultaneous exposures or sequential irradiations administered within 24 hours of each other.

In reviewing therapeutic studies specifically, it became clear that the term “combined modalities” is frequently applied to regimens in which different radiation types are administered with substantial time intervals between them—often separated by several days or even weeks—rather than in a truly sequential or simultaneous fashion. To maintain consistency with our definition of combinatorial exposures as involving either concurrent delivery or close-sequence administration (≤24 hours apart but for rare cases specifically annotated), such studies were generally excluded from this data set. Only protocols where different radiation modalities were applied in the same treatment timeframe—not exceeding 24 hours—were included, to avoid conflating simple sequential boosts with genuine mixed exposures.

The radiation types included in the search were near-infrared (NIR), visible (VIS), ultraviolet (UV), X-rays, gamma rays (γ), alpha radiation (α), beta radiation (β), protons (p), neutrons (n), and heavy ions. In addition, space-relevant mixed fields—such as high atomic number and energy (HZE) particles and simulated ion beam combinations (e.g., 5-ion or 33-ion beams)—were incorporated to reflect realistic space radiation exposures. Therapeutic modalities considered included conventional radiotherapy (RT), intensity-modulated radiation therapy (IMRT), boron neutron capture therapy (BNCT), gadolinium neutron capture therapy (GdNCT), carbon ion radiation therapy (CIRT), radionuclides therapy (RNT), proton therapy (PT), photodynamic therapy (PDT), photothermal therapy (PTT), near-infrared photoimmunotherapy (NIR-PIT), electron beam therapy (EBT), fast neutron therapy (FNT), megavoltage X-ray therapy (megavoltage XRT), and californium-252 neutron therapy (Cf-252 NT).

The methodological approach for the literature review involved several phases. Initially, databases and search engines—such as PubMed, Google Scholar, Semantic Scholar, bioRxiv, and Europe PMC—were searched manually using combinations of keywords, Boolean operators, and filters, covering publications from the 1960s to June 20, 2025 (the date of the final search for all databases). This temporal range emerged organically due to the historical spread of relevant studies and is indicative of the sustained and evolving nature of radiobiological research over multiple decades.

The majority of results were retrieved through PubMed, which served as the primary database for this review. To capture the diversity of radiation pairings and biological systems represented in the literature, over 50 manual keyword searches were conducted using tailored combinations of terms relevant to specific exposures, contexts, and biological endpoints. Representative full PubMed search strings included:

• ("alpha radiation"[tiab] OR "alpha particles"[MeSH Terms]) AND ("X-rays"[MeSH Terms] OR "X radiation"[tiab])
• ("simultaneous"[tiab] OR "sequential"[tiab]) AND ("irradiation"[tiab] OR "radiation"[MeSH Terms])
• "proton therapy"[tiab] AND "targeted radionuclide therapy"[tiab]
• "space radiation"[tiab] OR "simulated galactic cosmic rays"[tiab] OR "GCRsim"[tiab]

Similar search logic was applied across all other databases consulted. Filters used during searches included article type (journal articles) and language (English), with no species filters or field-specific restrictions applied. The “related articles” or “related results” features were also used to identify additional relevant publications. For radiation pairing categories initially represented by only one or very few reports, targeted follow-up searches were conducted to obtain a more representative sample. Despite these efforts, some combinations remain represented by a single entry; however, their inclusion was preferred over omitting those radiation combinations entirely from the review.

At least four reviewers independently screened titles and abstracts for eligibility. Discrepancies were resolved by re-evaluating studies against the predefined inclusion criteria and, where appropriate, through discussion of their relevance, representativeness, or scientific value in diversifying the data set—particularly with respect to biological systems or radiation combinations. The data extraction process involved up to three contributors. Data were collected manually into structured tables, with contributions verified and refined iteratively during the review process. Final consistency checks and consolidation were conducted by the lead author to ensure alignment with predefined data fields and classification categories. Following close reevaluation, 18 initially included reports were substituted with more relevant or representative studies based on refined eligibility interpretations. The final data set comprises 172 studies.

A master table was created, consisting of columns detailing the combined radiation types and doses, biological systems, biological endpoints, and effects recorded for each study. Abstracts and relevant sections of the selected publications were reviewed to extract key data, which were organized into structured entries corresponding to the table’s columns. Extracted biological outcomes included DNA damage, oxidative stress, cell viability, apoptosis, cell cycle arrest, gene expression changes, and, where applicable, organism-level responses.

In the final phase, the master table was split into separate tables, each corresponding to a specific combination of radiation types as studied across the selected publications. Rows were redistributed accordingly and reviewed manually. Within each table, rows were first sorted by radiation type combination, and then grouped by biological system. To facilitate identification of studies involving specific models within a given combined radiation context, biological systems were broadly categorized in the following order: human-derived systems, rodent models, other animals and plants, and microorganisms (including bacteria, viruses, and unicellular eukaryotes). The tables were then regrouped into three overarching categories: “Radiobiological Studies” (with the sub-categories Non-Ionizing–Ionizing, Non-Ionizing–Non-Ionizing, and Ionizing–Ionizing), “Therapeutic Studies,” and “Space Radiation Studies”. This restructuring resulted in five tables, which were inserted into the manuscript. The finalized data set then underwent analysis, processing, and discussion.

No data conversions or statistical imputation were applied during synthesis. A statistical synthesis of results was not undertaken, reflecting substantial variability in study designs, exposure protocols, and outcome measures. Given that the majority of included studies were mechanistic, conventional risk-of-bias instruments developed for clinical or preclinical investigations were not directly applicable across the entire data set. Methodological rigor was therefore appraised qualitatively, with attention to factors most relevant to the diverse experimental contexts represented. Effect sizes were not standardized, as most studies reported results qualitatively or using heterogeneous outcome metrics that precluded meaningful numerical comparison results were instead synthesized based on reported biological outcomes and qualitative trends. Potential reporting biases were not formally evaluated, and certainty of evidence was not rated, due to heterogeneity in radiation types, biological models, endpoints, and application contexts. All relevant data are reported within the manuscript; no additional data sets were generated or analyzed.

3. Results

A total of approximately 300 records were identified through database searches. After deduplication, screening, and eligibility assessment, 172 studies were included in the final review, as detailed in the PRISMA 2020 flow diagram (Figure 1). As outlined in the Methods, findings are presented narratively to reflect the diversity of study designs, biological systems, and outcome measures. Risk of bias and certainty of evidence were not formally assessed, in keeping with the qualitative synthesis approach.

In 18 cases, studies initially identified as eligible were replaced with equivalent reports more closely aligned with the predefined inclusion criteria. In accordance with PRISMA 2020 Item 16b, we include examples of studies that appeared to meet the criteria on initial review but were excluded after closer assessment. One such case is a report by Blanco et al., “Effects of Gamma and Electron Radiation on the Structural Integrity of Organic Molecules and Macromolecular Biomarkers Measured by Microarray Immunoassays and Their Astrobiological Implications”, which does describe combined irradiation effects on biological targets; however, these targets do not constitute true biological systems and the publication was therefore excluded [1]. Moreover, several therapeutic studies were excluded during eligibility reassessment due to technical mismatches with inclusion parameters. For example, a report by Aljabab et al., “A combined neutron and proton regimen for advanced salivary tumors: early clinical experience”, was excluded due to extended intervals between therapeutic modalities that far exceeded the temporal threshold set in our criteria [2]. Kumar et al., in “Senolytic agent ABT-263 mitigates low- and high-LET radiation-induced gastrointestinal cancer development in Apc^1638N/+ mice”, was similarly excluded upon reevaluation, as the study compared but did not combine radiation types, and therefore did not meet the fundamental inclusion requirement [3].

The studies which were eventually included span a wide range of radiation exposures, including combinations of ionizing and non-ionizing radiation, as well as high- and low-LET (Linear Energy Transfer) modalities. Given this diversity, a central aim of the review was to assess whether combined exposures yielded effects beyond those expected from individual radiation types. In this context, the term synergy is used to describe biological outcomes that exceed additive expectations based on single-exposure responses. Such outcomes include, for example, elevated levels of chromosomal aberrations, decreased cell survival, or gene expression changes that cannot be accounted for by a simple summation of effects from each radiation modality alone.

Quantitatively, synergy was inferred when the combined effect was statistically greater than the predicted additive response, or when modeled interaction terms (e.g., interaction index > 1) indicated non-linear enhancement. Studies in which the effect of combined exposure depended on irradiation order or the involvement of specific DNA repair pathways were also considered synergistic, provided that the observed biological damage exceeded what would be expected from the individual exposures alone.

The presented studies are summarized in five structured tables, each organized by radiation type and dose, biological system, assessed endpoints, and observed outcomes. The included summary tables provide an overview of study design and scope, while specific findings are detailed in the corresponding text sections.

3.1. Radiobiological Studies

3.1.1. Combinations of Non-Ionizing and Ionizing Radiation

Table 1 summarizes 17 experimental studies investigating combined exposures to non-ionizing and ionizing radiation, with a primary focus on ultraviolet (UV) radiation paired with X-rays, gamma rays, beta particles, or protons [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. The research spans human, mammalian, and microbial cell models, and examines endpoints such as chromosomal aberrations, clonogenic survival, DNA repair, and enzyme activity. Many of the included studies report synergistic effects. These outcomes often depended on factors such as cell cycle stage, radiation sequence, dose, and DNA repair competence. Selected examples are presented below to illustrate recurring patterns and mechanistic trends.

Among the included studies, one found no detectable synergy between UV and X-ray exposure in PHA-stimulated human lymphocytes in the G₁ phase, as dicentric chromosome yields were equivalent to those induced by X-rays alone [[4]. In contrast, another study using G₀-phase human peripheral blood lymphocytes reported approximately a two-fold increase in dicentric chromosome formation when UV and X-rays were administered within 30 seconds of each other, regardless of UV dose or exposure order [5].

Furthermore, one study reported that when UV preceded X-rays, the increase in dicentric chromosome formation remained stable across inter-exposure intervals up to 90 minutes; in contrast, when the order was reversed, the effect declined with a half-life of approximately 20 minutes [7]. Similarly, in V79 cells, UV followed by X-rays resulted in greater loss of clonogenic survival and repair capacity compared to the reverse sequence [8].

Beyond mammalian systems, synergistic interactions were also reported in microbial models. For instance, in bacteria, UV pretreatment sensitized Escherichia coli to X-ray–induced lethality, particularly in wild-type and uvr mutants. However, no synergy was detected in recA and recB mutants, while polA mutants showed impaired repair recovery after irradiation [9,10]. In yeast models, UV exposure prior to gamma irradiation led to more than 100-fold reductions in survival beyond additive predictions, and revealed strong synergy in Schizosaccharomyces pombe wild-type cells but not in recombination-deficient mutants [13]. UV pretreatment enhanced the lethality of incorporated ³²P decay in Salmonella, with the synergistic effect almost completely eliminated by photoreactivation [16]. Moreover, UV followed by proton irradiation caused a stronger reduction in bacterial survival than the reverse sequence [18]. Applied microbial studies further demonstrated that combined UV and gamma irradiation increased antifungal activity and mutagenesis in Bacillus velezensis strains evaluated through bioindustrial screening, with increased surfactin production and broader isoform profiles observed in selected mutants [15].

Notably, in a full-thickness human skin model, pre-irradiation with water-filtered infrared-A radiation (wIRA, a subtype of near-infrared radiation) prior to X-ray exposure resulted in delayed repair kinetics, with prolonged persistence of γH2AX and 53BP1 foci. Reduced fibroblast apoptosis and preserved tissue morphology were also observed following combined exposure [19].

3.1.2. Combinations of Non-Ionizing Radiation Types

Table 2 presents findings from 18 experimental studies investigating combined exposures involving non-ionizing radiation types, primarily various ultraviolet wavelengths in combination with visible light, near-infrared, or other UV subtypes. The studies span human, rodent, invertebrate, plant, and microbial biological models, and assess endpoints such as microbial inactivation, gene expression, oxidative stress, DNA damage and tumor incidence. While experimental systems and dose ranges vary, many studies report synergistic effects.

In E. coli, simultaneous UV-A and UV-B exposure produced approximately 100-fold greater inactivation than UV-B alone, mediated by s⁴U modifications in tRNA [26]. In parallel, human skin tissue models showed synergistic effects on photoaging biomarkers, oxidative stress, and gene expression following combined UV-B, UV-A, and near-infrared exposure, distinct from single or sequential treatments [22]. Furthermore, combined UV and visible light exposure in ragweed-allergic patients significantly inhibited allergen-induced wheal formation, whereas neither UV-A nor visible light alone produced this effect. The mixed exposure (mUV/VIS) suppressed mast cell–mediated responses in a dose-dependent manner, including at suberythematous doses [21].

Additional synergistic effects were observed in plant, invertebrate, and microbial systems. In tomato plants, UV-B and UV-C exposure increased disease resistance and antioxidant gene expression [25]. In marine invertebrates, long-term UV-A and UV-B irradiation reduced reproductive output and feeding capacity [24]. In environmental disinfection studies, complete microbial inactivation of Enterococcus faecalis and E. coli was achieved in synthetic water matrices using dual-UV systems, with synergy modulated by fluence and pH in certain setups [31]. Sequential UV-C exposure with KrCl excimer (222 nm) and low-pressure mercury lamps (254 nm) synergistically enhanced viral inactivation, with energy requirements influenced by total dose and exposure order [37].

3.1.3. Combinations of Ionizing Radiation Types

Table 3 provides an overview of 52 radiobiological studies investigating combinations of ionizing radiation types, primarily involving alpha particles, X-rays, gamma rays, protons, and neutrons. Most studies employed in vitro models, with additional in vivo and environmental systems, and explored endpoints such as clonogenic survival, DNA damage signaling, chromosomal aberrations, gene expression, inflammatory markers, and mutation rates. The biological responses varied widely depending on radiation quality, dose, sequence, and cell type. While synergistic effects were frequently reported—particularly in mixed alpha–X-ray and neutron–gamma exposures—several studies observed purely additive or even antagonistic outcomes. Select examples are outlined below to highlight key patterns in damage complexity, repair interference, and sequence dependence.

In human peripheral blood lymphocytes, combined alpha and X-ray exposure significantly increased complex chromosomal aberrations in a dose-dependent manner, with non-additive effects and a linear-quadratic response observed at higher mixed doses [43]. In U2OS osteosarcoma cells, co-exposure delayed decay of 53BP1 foci and prolonged ATM and p53 signaling, with the strongest effects at lower total doses [45]. Similar findings were reported for sequential alpha and gamma radiation, where alpha-first exposure produced larger and more persistent foci than the reverse sequence [55]. A related study in CHO-K1 cells observed delayed HPRT mutation induction and increased chromosomal aberrations after alpha–X-ray exposure, with effects persisting over multiple doublings in slow-growing subclones [54].

Mixed-exposure effects on clonogenic survival varied substantially by system and irradiation parameters. In V79 Chinese hamster lung fibroblasts, neutron–gamma co-exposure produced supra-additive reductions in survival, particularly under simultaneous delivery. Survival curves fitted better with quadratic models, indicating interaction between damage types [78]. In rat lung epithelial cells (LECs), combined alpha (1 Gy) and X-ray exposure eliminated the survival curve shoulder and reduced clonogenic survival beyond additive expectations. Micronuclei frequencies were also elevated under combined exposure, consistent with non-linear interaction effects [53]. In contrast, a study in Chinese hamster ovary (AA8) cells using mixed alpha and X-ray beams reported no evidence of synergy, with survival data aligning closely with predictions from mathematical additivity models [51].

At the transcriptional level, combined radiation exposures modulated key stress and damage response genes across multiple systems. In human lymphocytes, combined alpha–X-ray exposure increased FDXR, GADD45A, and MDM2 expression beyond levels induced by alpha radiation alone in most donors, with synergy confirmed in 3 out of 4 cases using envelope-of-additivity analysis. Furthermore, ATM inhibition reduced this response, implicating checkpoint signaling [42]. Neutron–photon mixtures elicited strong transcriptomic effects even at low neutron fractions. In murine models, as little as 5% neutron contribution suppressed EIF2/mTOR signaling and ribosomal protein expression—alterations not observed with X-rays alone [62]. In human peripheral blood, increasing neutron percentages led to enhanced TP53 signaling and broader immune dysregulation [61]. Other studies reported dose-dependent modulation of BAX, DDB2, and FDXR expression following neutron–gamma co-exposure [72].

Beyond transcriptional responses, several studies show that mixed-field irradiation can also trigger broader cellular and physiological adaptations. In murine blood cells, neutron–gamma exposure altered membrane architecture and lectin-binding patterns, with lymphocytes and platelets showing the most marked ultrastructural remodeling [80]. In separate mouse models, combined exposures impaired hippocampus-dependent memory and shifted neuroimmune profiles toward an anti-inflammatory state [81].

A rare entry in the data set examined neutron–proton co-exposure in human breast cancer cell lines, offering insight into mixed-beam effects on cancer stem cell (CSC) populations. The response varied by cell line: CSC fractions declined additively in MCF-7 cells but antagonistically in MDA-MB-231, with no significant changes in canonical stemness gene expression [86]. This study was among the few to examine neutron–proton co-exposure, providing rare data on beam combinations with potential relevance to radiation protection and therapy

Additional underrepresented combinations include alpha–beta inhalation in rats, which were found to elicit additive impairments in pulmonary function [60], and mixed radionuclide exposure in plants, where barley grown in contaminated soil accumulated mutations at rates exceeding those predicted by dose alone [58]. Though differing in species and context, these models replicate real-world exposure scenarios such as internal contamination and chronic environmental irradiation, and demonstrated effects exceeding predictions based on dose alone.

3.2. Therapeutic Studies

Table 4 outlines 37 clinical, preclinical, and in vitro studies evaluating therapeutic applications of mixed radiation modalities. The reported outcomes reflect the influence of beam combinations, sequencing, timing, and model system, with examples spanning conventional photons, protons, neutrons, heavy ions, and photodynamic approaches.

Clinical investigations of mixed proton–photon regimens have demonstrated favorable outcomes across several tumor types. In supratentorial glioblastoma, a combined modality approach using daily photon irradiation with a proton concomitant boost delivered more than 6 hours later achieved a median survival of 21.6 months and manageable toxicity, with occasional late leukoencephalopathy [91]. Similarly, in stage II–IV oropharyngeal squamous cell carcinoma, a regimen incorporating photons and a proton boost produced a 5-year locoregional control rate of 84%, although acute mucosal toxicity and grade 3 late effects were observed in some patients [92]. For skull base and cervical spine chordomas and chondrosarcomas, sequential photon–proton treatments resulted in excellent long-term control for chondrosarcomas (94% at 10 years) and moderate control for chordomas, with late complications including temporal lobe injury and endocrinopathy [93].

In Chinese hamster fibrosarcoma cells, sequential carbon-to-proton irradiation with a 45% carbon contribution induced significant synergy, while reversing the order diminished the effect or led to antagonism [94]. In a related study using mixed ¹⁶O/²⁸Si/proton beams, greater high-LET contributions suppressed survival more effectively, particularly when delivered before protons. Notably, a proton-to-high-LET sequence permitted partial recovery, indicating an order-dependent effect [97].

Several studies evaluated neutron-based combinations. In a murine study, neutron–proton sequencing influenced both toxicity and tumor outcomes in solid Ehrlich ascites carcinoma. Neutron-first exposures exacerbated skin damage and reduced survival, while the reverse sequence was better tolerated [98]. These findings were echoed in vitro, where neutron-before-proton combinations consistently produced greater reductions in clonogenic survival than the opposite order, indicating sequence-dependent efficacy of mixed-beam regimens [99]. In clinical settings, mixed neutron–photon protocols have been investigated in patients with malignant gliomas, where neutron boosts were delivered within minutes or hours of photon irradiation. One such study administered a neutron boost 5–20 minutes before whole brain photon delivery and reported markedly prolonged survival in anaplastic astrocytoma but not glioblastoma [118]. In a separate randomized trial, twice-weekly neutron boosts were given within 3 hours of photon therapy, achieving frequent tumor sterilization at autopsy but also substantial radiation injury [119].

Other investigations have combined external beam therapy with biologically targeted or nanotechnology-enhanced modalities. In mouse xenograft models, combining proton irradiation with ¹⁷⁷Lu-labeled targeted radionuclides produced additive or synergistic tumor suppression depending on the tumor model [95]. Similarly, preclinical work combining low-energy X-rays with ¹⁷⁷Lu-PSMA-617 radionuclide therapy in prostate cancer xenografts prolonged tumor doubling time and increased median survival compared to external beam radiation alone [96]. Photodynamic therapy (PDT) integrated with photon radiotherapy (RT) has demonstrated synergistic antitumor effects in selected preclinical systems. For example, studies in bladder cancer organoids showed enhanced cell death and immune migration [102], while pancreatic cancer spheroids and co-cultures exhibited increased apoptosis, DNA damage, and reduced viability [104]. In breast cancer cells, PDT with indocyanine green (ICG) and low-dose X-rays substantially increased cell killing compared to monotherapies [106]. Additionally, X-ray-triggered UV-C emission from nanoscintillators enhanced cytotoxicity and G2/M arrest in lung cancer spheroids [107] and potentiated CPD DNA damage and clonogenic suppression in UV-sensitive fibroblasts [108]. In early-phase clinical studies of cervical cancer, initiating HDR brachytherapy during pelvic external beam radiotherapy resulted in approximately 70–75% tumor volume reduction by the first brachytherapy fraction, supporting the feasibility of integrated regimens [110].

One of the included studies investigated a combined photon–electron beam technique for breast irradiation. Seven patients with early-stage breast cancer underwent accelerated partial breast irradiation following lumpectomy, receiving a total dose of 38.5 Gy in 10 fractions over five days. Treatment consisted of both modulated electron beams (9–15 MeV) and intensity-modulated photon fields (6 MV) delivered within each fraction. The reported results showed adequate target coverage, lower radiation exposure to the ipsilateral lung and heart compared to IMRT alone, no grade 2 or higher acute toxicity, and favorable cosmetic outcomes in the majority of cases [111].

Several studies investigated boron neutron capture therapy (BNCT), which involves high-LET thermal, epithermal, or fast neutrons combined with low-LET gamma or X-rays. In vitro mixed-field exposures have demonstrated additive effects on survival endpoints, for example in alpha–gamma co-irradiation models of V79-4 Chinese hamster cells [123]. In contrast, neutron–gamma exposures have been associated with persistent and spatially clustered DNA damage, with larger and more complex foci compared to gamma irradiation alone, consistent with increased damage complexity and altered repair dynamics [122]. Notably, one study reported a strong synergistic effect on cell killing when alpha particles and X-rays were delivered simultaneously at higher alpha doses, suggesting that alpha exposure can impair DNA repair from low-LET X-rays [124]. Furthermore, BNCT-associated mixed-beam exposures caused more pronounced effects in wild-type than in repair-deficient cell lines [125]. A related investigation demonstrated that thermal neutrons generated during proton beam therapy could also be harnessed to trigger boron neutron capture reactions, significantly enhancing cell killing in vitro [121]. More recently, a combined boron and gadolinium neutron capture therapy approach was tested, delivering both high-LET alpha particles and gamma rays in a single neutron exposure. In preclinical head-and-neck tumors, this approach achieved nearly complete tumor eradication and strong suppression of recurrence biomarkers [126].

3.3. Space Radiation Studies

Table 5 presents 48 studies reporting biological effects of combined ionizing radiation exposures relevant to spaceflight. These include sequential exposures involving 2 to 33 distinct ion beams, as well as complex mixed-field exposures that mimic space radiation —particularly galactic cosmic radiation (GCR), simulated using GCRsim models or simplified-beam versions known as Smf-GCR. The studies include in vitro models, animal experiments, and one spaceflight case (NASA Twins Study). Reported endpoints span DNA damage, chromosomal aberrations, cellular transformation, neurocognitive and behavioral assessments, immune and cardiovascular outcomes, and tissue-specific effects. Observed effects varied with exposure parameters—such as sequence, timing, and composition—as well as biological factors including sex, tissue type, and model system.

Many studies using GCRsim beams reported dose- and sex-specific deficits in short-term memory, spatial learning, and cognitive flexibility at total doses of ~0.5–1 Gy. In a 2-ion study, these effects were linked to dendritic simplification, reduced mushroom spine density, and altered expression of GluR1, NR2A, and Synapsin-1 [132]. A related study reported similar structural changes and SNP alterations, though synaptic proteins were not assessed [133]. In another 5-beam study, male-specific cognitive deficits were causally linked to microglial activation and hippocampal remodeling, with early blood monocytes indicating late-stage impairment [142].

In many cases, low-dose GCRsim exposure was found to alter complex behaviors in a sex-dependent manner. For instance, in mice, females showed increased grooming and burrowing activity at 15–50 cGy, with nest-building performance varying by dose and sex, despite intact sensorimotor function [145]. In a similar experiment, female rats displayed increased risk-taking after 10 cGy, while males showed slower decision speeds, both normalizing by 90 days [149]. In a touchscreen-based task, 10 cGy impaired switch accuracy in females (~20%), increased perseverative errors, and reduced training success under cognitive load [151].

Chronic and acute exposures impaired memory updating and object recognition in a sex-dependent manner. Long-term potentiation (LTP) was reduced, and changes in postsynaptic density and axonal myelination were observed in mice [162]. Complementary touchscreen-based testing showed that male mice developed attentional deficits and slower response times, alongside disrupted prefrontal dopamine signaling and altered neurotransmitter network organization [163]. One study using 6-ion exposures found that pharmacological HDAC3 inhibition reversed LTP deficits and restored behavioral performance, indicating that epigenetic modulation can mitigate GCR-induced cognitive impairments [160].

Parallel investigations assessed cancer risk and tissue remodeling under space-relevant mixed radiation. In K-ras^LA1 mice, chronic 33-ion GCRsim exposure increased lung adenocarcinoma incidence, an effect further enhanced by a delayed neutron dose [165]. In C57Bl/6J mice, exposure to a simplified five-ion GCR simulation impaired novel object recognition and altered brain connectivity, effects that were partially mitigated by prophylactic amifostine treatment [156]. Mammary gland studies in Apc^Min/+ mice showed increased ductal hyperplasia and overgrowth, along with elevated expression of ERα, ERRα, and SPP1—markers associated with estrogen signaling and tumor development [161,168].

In primary human fibroblasts, sequential exposure to protons followed by titanium or iron ions with a 15-minute interval produced a marked synergistic increase in anchorage-independent growth, even at low proton doses. This effect was not observed with reversed ion order or same-ion split doses [130]. This pattern was reinforced in neonatal fibroblasts, where transformation rates were up to three times higher than additive predictions when protons were followed by HZE ions within a 2.5-minute to 6-hour window, but not when the order was reversed [131]. In human mammary epithelial cells, chromosome aberration frequency was highest when iron ions were delivered 30 minutes after protons. Combined exposure produced more damage than the predicted additive response, with both intra- and inter-chromosomal exchanges elevated at this interval [128].

Radiation-induced cardiovascular and systemic effects were documented across multiple models. In WAG/RijCmcr rats, whole-body exposure to a mixed field of protons, silicon, and iron ions led to perivascular cardiac fibrosis, increased systolic blood pressure, and infiltration of CD68+ macrophages in the heart and kidneys—effects not observed with single-ion exposures [137]. In BALB/c and CD1 mice, 5-ion GCRsim caused sex- and strain-specific changes in cardiac structure, including increased collagen deposition, altered capillary density, and modulation of immune markers such as CD2 and TLR4 [144]. To mimic the persistent radiation-induced biological effects observed in space exposure scenarios, another study using a myocardial infarction model administered proton and iron irradiation over a 48-hour interval—exceeding the ≤24-hour threshold used in almost every study reviewed here—which significantly influenced myocardial fibrosis and infarct size [134]. Peripheral immune alterations included changes in leukocyte profiles and phagocytic activity, with notable sex-specific transcriptional shifts [140].

Studies also implicated the gut microbiome as a sensitive and interactive component. In several mouse models, microbiota composition changed after GCRsim in a sex- and dose-dependent manner and correlated with cognitive, affective, and neuropathological outcomes [135,148,159].

Other systemic interactions included viral reactivation, endocrine modulation, and altered intercellular communication. In latently infected human myeloblasts, GCRsim and high-LET ions reactivated CMV and induced viral gene expression in a dose- and LET-dependent fashion [157]. In co-culture systems, proton pre-exposure suppressed the bystander response to iron ions in human fibroblasts, underscoring the impact of prior radiation history on intercellular signaling dynamics [129].

In the NASA Twins Study, 340 days of spaceflight—including exposure to approximately 76 mGy of galactic cosmic radiation—resulted in persistent chromosomal inversions, telomere elongation followed by rapid shortening, immune dysregulation, cognitive decline, and altered microbiome composition. Multi-omic analyses showed that many of these effects persisted post-flight and mirrored findings from rodent GCR models [175].

Several studies have tested countermeasures against GCR-induced effects. Low-dose X-ray pretreatment improved viability and reduced ROS in cardiomyoblasts exposed to GCRsim [154]. The antioxidant CDDO-EA mitigated cognitive deficits and restored neurogenesis in female mice [166], though its effects on social behavior were variable [[167]. Furthermore, HDAC3 inhibition was shown to reverse synaptic plasticity impairments [160]. In contrast, aspirin failed to protect and worsened cognitive performance in control animals [171].

4. Discussion

The systematic collection, classification, and synthesis of studies on combined irradiation of biological systems conducted in this review provided critical insights into the current state of the field. The research landscape is consistently structured around three main contexts: Radiobiology, Therapeutic Applications, and Space Radiation Research. This tripartite framework reflects fundamentally distinct objectives—mechanistic elucidation, clinical application, and astronaut health risk mitigation, respectively.

4.1. Mechanistic Basis of Mixed Radiation Effects

Radiobiological studies have consistently revealed that combinations of ionizing and non-ionizing radiation produce distinctive biological effects compared to single-modality exposures. These effects are shaped by variables such as radiation type, dose, timing, and cellular context. As summarized in Figure 2, such interactions often lead to complex DNA damage, saturation of repair capacity, and the emergence of genomic instability.

The figure was created using Biorender, available at https://app.biorender.com/illustrations.

4.1.1. Exposure Sequence Dependence in Mixed Radiation Exposures

Evidence from a wide range of biological systems—including bacteria, primary human cells, established mammalian lines, and whole organisms—demonstrates that the impact of sequential mixed radiation exposures extends beyond simple dose accumulation, showing that timing and cellular context can be critical determinants of outcome. In combinations of non-ionizing and ionizing radiation, many studies of UV–ionizing pairings either did not systematically assess the role of exposure sequence or reported it as having limited impact, yet several investigations have clearly shown that the temporal order of exposures can determine whether damage responses remain additive or become strongly synergistic. For example, in human lymphocytes, UV irradiation preceding X-rays resulted in a stable two-fold increase in chromosome aberrations, whereas reversing the order caused a rapid decline in synergy [7]. Similarly, UV pre-exposure sensitized E. coli to proton irradiation, while the reverse sequence did not produce synergy [18], and in Micrococcus radiophilus, UV pretreatment yielded pronounced synergistic killing, contrasting with additive effects when gamma rays were applied first [14].

Expanding on this pattern, similar effects have been observed across varied biological models and irradiation conditions. Sequential exposure of MS2 bacteriophage to different UV wavelengths led to significantly greater inactivation when low-wavelength UV preceded higher-wavelength UV [[37]. In U2OS cells, alpha radiation delivered before gamma irradiation induced more persistent DNA repair foci and chromatin remodeling [56]. In Chinese hamster fibrosarcoma cells, carbon ion priming heightened the cytotoxic impact of subsequent proton irradiation [94], while neutron–proton combinations proved most effective when neutrons were applied first [99]. More recent studies modeling space radiation environments also illustrate this phenomenon: in murine lung tissue, delivering protons prior to silicon ions markedly increased premalignant lesions [136], and in primary human fibroblasts, proton priming before HZE ion exposure tripled transformation markers compared to the reverse sequence [131].

This set of examples helps demonstrate that sequence-dependent interactions are neither confined to any one radiation pairing nor restricted to a single biological context. Instead, they represent a broadly observed effect that likely arises from the interplay between DNA damage complexity, cellular stress responses, and the inherent repair competence of each system.

4.1.2. DNA Repair Dependencies, Lesion Interactions, and Temporal Modulation

Microbial systems have been especially valuable for clarifying the molecular basis of these interactions, as they allow precise dissection of DNA repair pathways through genetic manipulation. In E. coli, synergistic lethality from combined UV and X-ray exposures was observed in wild-type and polA mutants but abolished in recA, recB, and recC mutants, implicating homologous recombination (Type III repair) as a major contributor to synergy. The partial effect seen in polA mutants suggests that base excision or Type II repair may modulate the response as well, though they are not sufficient on their own [9,10]. Further work using B/r and Bs-1 strains confirmed that UV pretreatment interferes with the repair of strand breaks: synergistic X-ray sensitization occurred only in wild-type B/r cells and was abolished when DNA metabolism was perturbed by 5-bromouracil substitution or purine starvation—conditions known to inhibit DNA repair [11]. In human leukocytes, combined UV and X-ray exposure led to greater chromosome damage in both healthy and Down syndrome donors, with diminished DNA repair synthesis observed in the latter [6]. Overlapping repair pathways may therefore mediate synergistic outcomes, with these effects being strongly influenced by cellular repair competence.

Critically, the temporal arrangement of UV and ionizing radiation exposures emerged as a key determinant of synergistic responses in mammalian cells. In peripheral human lymphocytes, a substantial two-fold increase in dicentric chromosome yield was consistently observed when UV irradiation preceded X-rays—even when inter-exposure intervals extended up to 90 minutes [7]. This stability indicates that UV-induced lesions can persist long enough to interfere with the repair of subsequent ionizing damage, thereby elevating the probability of chromosome aberration formation. In contrast, reversing the order of exposure led to a rapid decay in the synergistic effect, with a measured half-life of approximately 20 minutes. This decay strongly implicates short-lived intermediates—such as unrepaired single-strand breaks or transient chromatin alterations—in mediating the synergy when X-rays are delivered first. The interaction between lesions was therefore found to be highly time-sensitive, with DNA repair kinetics playing a central role in shaping interactive outcomes. Consistent with this observation, studies in murine intestinal crypts showed that mixed neutron and X-ray irradiation produced non-additive effects when delivered within a few hours of each other, but fully additive outcomes when sufficient time was allowed for sublethal damage repair—supporting the importance of lesion recovery windows in determining synergy or independence [69].

A key takeaway is that temporal parameters—such as lesion persistence, inter-exposure intervals, and the kinetics of repair pathway engagement—can decisively influence whether interactions are synergistic, additive, or even antagonistic. While short inter-exposure intervals may allow persistent lesions or intermediates from the first radiation to interfere with the cellular response to the second, longer delays may permit resolution, thereby diminishing interactive effects. For instance, pretreatment with water-filtered near-infrared radiation before X-ray exposure was reported to delay DNA damage repair while reducing apoptosis in full-thickness skin models, suggesting that certain non-ionizing modalities might help mitigate harmful effects of ionizing radiation through modulation of stress signaling pathways [19].

4.1.3. Cell Cycle-Specific Interaction Effects

Moreover, this temporal dependence aligns with cell cycle–specific differences in DNA repair dynamics. In G₀-phase lymphocytes exposed to a combination of UV radiation and X-rays, synergy was consistently observed, whereas in G₁-phase cells, no increase beyond additive expectations was detected [4]. This shift likely reflects the relative abundance and efficiency of lesion-processing pathways between quiescent and actively cycling cells. Studies in human lymphocytes have further elucidated how cell cycle stage and lesion persistence shape interactive outcomes. More experiments evaluating UV and X-ray co-exposures in quiescent (G₀-phase) and stimulated (G₁-phase) lymphocytes revealed that the presence or absence of active repair processes fundamentally alters the response to mixed radiation. In G₀-phase cells, UV-C irradiation administered immediately before X-rays consistently produced a two-fold increase in dicentric chromosome formation relative to X-rays alone [5,7]. In contrast, G₁-phase lymphocytes showed no evidence of synergy, with combined exposures yielding only chromatid-type aberrations typical of UV damage and dicentric frequencies indistinguishable from X-rays alone, suggesting that progression into G₁ allows for repairing or processing lesions before they interact [4]. This difference indicates that the cell’s position in the cycle determines whether damage responses remain compartmentalized or converge to produce more complex chromosomal changes.

Cell cycle–specific effects have been widely observed in mixed radiation studies. In synchronized V79 cells irradiated in late S-phase, sequential exposure to high-LET particles followed by X-rays produced marked synergism, even though this phase was relatively resistant to either radiation alone, indicating phase-dependent susceptibility to combined damage [89]. Analysis of sequential exposures across defined stages showed the greatest interaction in late S-phase, with smaller effects at the G₁/S border and in G₂, consistent with variations in repair capacity during the cycle [88]. In plateau-phase V79 cells—comprising a predominantly G₁ population—mixed neutron–gamma exposures yielded survival curve modifications closely resembling those produced by postirradiation treatment with β-araA, a DNA polymerase inhibitor that fixes potentially lethal damage by inhibiting post-replication repair; results indicated that the interaction effect in this context reflects repair constraints characteristic of G₁ [83]. In a different context, combined UV-emitting nanoparticles and ionizing radiation in 3D lung cancer spheroids enhanced cytotoxicity through apoptosis, necrosis, and persistent G₂/M arrest, illustrating that mixed exposures can exert phase-specific effects by overwhelming DNA repair pathways active during late cell cycle stages [107].

4.1.4. Mechanistic Insights from Non-Ionizing Radiation Combinations

In non-ionizing radiation combinations, synergistic effects have been observed across both environmental and biological systems, often involving complementary mechanisms of photodamage. In E. coli, simultaneous UV-A and UV-B exposure resulted in a nearly 100-fold increase in inactivation compared to UV-B alone, driven by UV-A absorption through thiouridine (s⁴U) residues in tRNA, which impaired translation and thereby reduced the cell’s capacity to repair subsequent DNA damage [26]. In human skin models, co-exposure to UV-A, UV-B, and near-infrared radiation produced a distinct biomolecular response characterized by heightened oxidative stress, elevated MMP-1 expression, and activation of MAPK signaling—effects not reproduced by individual or sequential exposures, suggesting wavelength-dependent crosstalk between damage and repair pathways [22]. This pattern implies that when photoreactive wavelengths converge, they can engage parallel cellular sensors and signaling cascades in ways that transcend mere summation, effectively reprogramming both acute stress responses and longer-term adaptations. In microbial systems, sequential UV-A and UV-C exposure produced persistent translational arrest and synergistic inactivation in wild-type E. coli, an effect absent in strains lacking thiI-dependent tRNA modifications, highlighting the role of non-DNA targets in modulating radiation sensitivity [27].

In a human skin model of allergen challenge, mixed ultraviolet and visible light (mUV/VIS) exposure produced dose-dependent suppression of mast cell–mediated wheal formation at suberythematous doses—an effect not seen with UV-A or visible light alone. This inhibitory response was attributed to the combined action of different wavelengths on histamine release from cutaneous mast cells, suggesting that such protocols may achieve immunomodulation with lower UVB doses, thereby reducing phototoxicity risks while retaining efficacy with possible translational therapeutic applications [21]. A key deduction from these findings is that synergy in non-ionizing radiation exposures often reflects the combined engagement of diverse cellular targets and signaling processes, which together generate biological effects that differ qualitatively from those induced by single-wavelength exposures.

4.1.5. Mechanisms and Functional Consequences of Combined Ionizing Radiation Exposure

Mechanistic insights from combinations of ionizing radiation types reveal that synergy frequently stems from the intersection of distinct lesion types and the breakdown of repair coordination. When alpha particles are combined with X-ray, the resulting DNA damage is not merely greater in magnitude but also altered in quality—evidenced by slower resolution of DNA repair foci, complex chromosomal aberrations, and sustained ATM and p53 signaling [43,45]. Supporting this, a study in CHO-K1 cells found that co-exposure to alpha and X-rays led to delayed HPRT mutations and increased chromosomal aberrations in slow-growing subclones, with mutation frequencies remaining elevated even after 23 doublings [54]. Overall, the evidence indicates that mixed fields can induce persistent genomic instability and engage stress response pathways more robustly than single modalities. Supra-additive effects observed even at modest combined doses suggest that repair mechanisms may be overwhelmed under conditions where simple additivity would otherwise be expected.

Transcriptional reprogramming offers insight into how cells perceive and prioritize competing damage signals. Co-exposures such as alpha–X-ray and neutron–photon combinations induced distinct expression signatures, including upregulation of DNA damage response genes (e.g., FDXR, GADD45A, MDM2) and suppression of translation-related pathways such as EIF2/mTOR signaling [42,62]. Combined neutron and gamma irradiation further engaged a broader spectrum of transcriptional alterations, with more differentially expressed genes and enriched signaling and metabolic pathways compared to gamma rays alone [72]. Co-exposure was associated with activation of apoptosis and DNA repair programs and dose-dependent shifts in key markers, supporting their potential utility as biodosimetric indicators. Neutron–photon mixtures also triggered enhanced TP53 activity and immune dysregulation as neutron fractions increased [61], suggesting multilayered stress integration beyond additive effects. Importantly, these effects occur even in the absence of cell death, indicating that transcriptional signatures capturing DNA repair engagement, metabolic disruption, and immune modulation may serve as early predictors of synergistic stress—crucial for biodosimetry and mechanistic understanding of mixed-field exposures.

Clonogenic survival data link molecular changes to functional outcomes. Supra-additive losses in colony formation under combined neutron–gamma or alpha–X-ray exposure reflect cumulative disruptions to proliferation and genome stability [39,53,54,56,75,78,87]. Sequential exposure studies have further shown that pre-irradiation with alpha particles can markedly reduce the shoulder of subsequent X-ray survival curves without appreciably changing slope within the studied dose range, indicating persistent sublethal damage capable of interacting with later insults [52]. Moreover, experiments in yeast have demonstrated clear synergistic effects when alpha and gamma radiation are applied simultaneously, with mutation frequencies exceeding additive expectations by over 30% [57]. An important clarification to be made is that not all systems or experimental conditions yield detectable synergy; in AA8 cells, survival curves under simultaneous mixed-beam exposure were consistent with additive predictions, suggesting that under these specific parameters, combined irradiation did not enhance clonogenic loss [51]. This variability reinforces the role of cell-specific repair competence and exposure sequence in shaping whether synergy manifests—an essential consideration in therapeutic planning and radioprotective modelling.

4.1.6. Multiscale Biological Effects and Uncommon Exposure Scenarios

Beyond nuclear targets, mixed-field exposures have been found to affect broader cellular structures and physiological systems, including membrane organization in blood cells [80], neuroimmune processes in the central nervous system [81], systemic metabolic responses reflected in serum fluorescence changes [[79], and pro-inflammatory lipidomic alterations indicating dysregulated lipid metabolism [85]. Such effects demonstrate that combined radiation can disrupt multiple levels of biological organization, which has important implications for risk assessment in complex exposure scenarios, including space-flight and radiological emergencies.

Interestingly, even rarely tested combinations such as neutron–proton co-exposure have revealed subtype-specific effects on cancer stem cell populations, indicating that such combinations may produce differential responses not observed with individual modalities [86]. Likewise, environmental models involving complex or atypical exposure scenarios—such as mixed radionuclide contamination in plants or alpha–beta inhalation in rodents—demonstrate that chronic, low-dose synergy can lead to mutation accumulation and functional tissue impairment over time [58,60]. It thus becomes evident that radiation synergy is not merely a byproduct of experimental design but a genuine biological phenomenon that occurs in real-world contexts. As such, mixed-field effects must be integrated into predictive models of environmental risk, long-term health outcomes, and radiological protection—especially in scenarios dominated by chronic, low-dose exposures.

Radiobiological findings discussed here indicate that, under defined conditions, combined radiation exposures can lead to amplified or qualitatively distinct biological outcomes, driven by interactions at the levels of DNA damage formation, repair kinetics, and stress response signaling. A deeper understanding of these dynamics is essential for refining risk assessment models and for effectively harnessing combinatorial effects in both therapeutic interventions and protective strategies in space exploration.

4.2. Determinants and Mechanisms of Synergy

Synergy—defined in this review as a biological effect exceeding the sum of individual radiation exposures and referred to elsewhere in this review as “supra-additive” or “non-additive” outcomes—typically emerges not from simple dose accumulation but from the interaction of distinct stressors in ways that challenge or exceed a cell’s adaptive capacity. Across the combinations examined in Section 4.1, synergistic outcomes are linked to circumstances in which early damage persists or leaves molecular “footprints” (e.g., persistent γH2AX/53BP1 foci, chromatin compaction changes, or mitochondrial ROS carryover) that alter the processing of subsequent insults. These conditions are often shaped by exposure sequence, radiation quality, dose rate, oxygenation status, and the repair competence and cell-cycle position of the responding system, with cell type–specific repair capacity also strongly influencing whether synergy manifests.

While in many cases mixed-field outcomes are found to be additive or even antagonistic—sometimes due to adaptive responses, checkpoint-mediated slowing, or induced buffering—synergy appears to be more likely when interacting lesions or stress signals converge on repair- or signaling-limited states, such as saturation of homologous recombination capacity or checkpoint arrest without full repair. At the molecular level, co-exposures have been shown to shift transcriptional and post-translational programs and repair pathway priorities in ways that can be qualitatively distinct from single-modality responses—reconfiguring metabolic balance and immune signaling. Such emergent patterns are often not simple amplifications of known responses but reflect the integration of disparate inputs across damage sensing, checkpoint coordination, and recovery pathways.

Recognizing synergy as a product of interaction rather than accumulation is essential for predictive modeling, since its occurrence depends on a defined set of parameters—dose, radiation quality, timing, and biological context—that determine whether damage responses remain independent or are re-shaped into outcomes exceeding additive expectations.

4.3. Therapeutic and Protective Strategies

Clinical studies indicate that combining different radiation types often enhances therapeutic effects beyond what would be expected from single modalities, albeit not always without collateral adversities. For example, proton–photon regimens have yielded promising control rates in glioblastoma, oropharyngeal, and skull base tumors, but also revealed significant late complications such as leukoencephalopathy, mucosal toxicity, and temporal lobe injury [91,92,93]. Other clinical studies, combining neutrons and photons, reported improved tumor control and survival in anaplastic astrocytoma, albeit with frequent radiation injury [118,119], again reflecting the observed balance between therapeutic benefit and treatment-related risk. Harnessing the advantages of combined radiations for therapeutic aims therefore requires continual refinement of planning, both dosimetric and methodological, as well as reinforcement through preclinical and radiobiological experimentation. In this context, it is worth noting that the reported favorable outcomes of partial breast irradiation using photon–electron beams were made possible by prior high-precision, Monte Carlo–based planning and overall careful treatment design [111]. Ultimately, the therapeutic value of mixed-beam strategies lies in their capacity to exploit complementary physical and biological effects, but their clinical success depends on anticipating adverse effects and integrating radiobiological insight into treatment protocol.

Beyond external beams, hybrid approaches that merge radiation with nanotechnology or molecular targeting are being explored. PDT combined with photon RT was shown to enhance tumor killing in multiple models [102,103,104,105,106]. Radiosensitization via targeted radiopharmaceuticals or nanoscintillator-triggered UV damage suggests that combinatorial designs can exploit biological vulnerabilities in novel ways to boost therapeutic efficacy by enhancing localized radiation delivery and increasing tumor-specific effects [95,96,107,108]. Further combinations involving targeted modalities, such as HDR brachytherapy in conjunction with external beam radiation therapy (EBRT), have shown promise in certain contexts, providing more precise tumor targeting, enhanced tumor shrinkage, and reduced normal tissue exposure [110].

In medical, environmental, and spaceflight settings involving combined radiation exposures, protective strategies are essential to reduce associated risks. These methods have been assessed in radiation pairings across the spectrum, aiming at attenuating damage through supplementary protective agents or at promoting adaptive responses by use of a preceding irradiation. The latter methodological approach is of specific interest in the scope of this review. For instance, in medical applications, techniques such as photobiomodulation, which use pre-irradiation with therapeutic low-intensity light, exemplify how such interventions can modulate adaptive cellular responses and influence defense systems, potentially activating protective signaling pathways and supporting tissue preservation [19]. Similarly, pre-exposure to low-intensity laser irradiation in rats has shown radioprotective effects by enhancing antioxidant defense and supporting hematological recovery [20]. Furthermore, one example of low-dose X-ray pre-exposure in the context of space radiation research has illustrated how adaptive responses can be triggered to improve radiation tolerance in cellular models, providing further insight into radiation’s potential adaptive effects [154]. Overall, the takeaway is that carefully tailored radiation strategies, informed by the timing, dose, and type of exposure, can create new paths for both amplifying therapeutic efficacy and enhancing environmental and space radiation protection, provided these factors are precisely controlled. Ongoing research is essential to delineate the mechanisms and optimize protocols for safe and effective implementation.

Altogether, mixed-field approaches offer significant promise for increasing treatment specificity, minimizing collateral damage, and revealing tumor-selective response windows. Future strategies may benefit from personalizing beam selection and delivery sequence based on tumor type, molecular profile, and repair capacity. In parallel, the development of radioprotective interventions—such as photobiomodulation or adaptive preconditioning—may help safeguard healthy tissues across a range of exposure contexts, from clinical radiotherapy to occupational, environmental, and spaceflight scenarios. As mechanistic understanding of these interactions deepens, such approaches could contribute to comprehensive radiation management strategies.

4.4. Space and Environmental Risk Contexts

Space radiation research increasingly shows that combined-field exposures, especially Galactic Cosmic Ray simulations blending protons with high-LET ions, can produce complex biological responses that defy predictions based on single-beam or purely dose-proportional models. Rather than scaling uniformly with total dose, these effects arise from interactions between radiation qualities and exposure sequences, with even low-dose mixed fields reported to disrupt such diverse systems and functions as cognition, synaptic integrity, and behavioral performance in ways often found to differ by sex and genetic background [135,163]. Similar order-dependent sensitivities have emerged in space radiobiological models, where the timing and sequence of proton and high-LET ion irradiation were found to determine not only the magnitude of neoplastic transformation in vitro [130,131] but also, in animal studies, whether tumors develop and how aggressively they progress [136]. These deductions strongly support the need for protective strategies that take into account the interplay of radiation quality, sequence, and timing.

In space-related research, chronic exposure has been identified as a key factor influencing biological risk. For instance, in K-ras^LA1 mice, chronic GCRsim regimens were shown to increase lung tumor incidence more than acute exposures [165]. However, this distinction is not limited to typical radiobiological endpoints; several neurobehavioral studies have deliberately contrasted chronic and acute dosing schedules to uncover differences in the persistence, scope, and nature of the effects. In prefrontal cortex function, chronic GCRsim exposure was linked to sustained deficits in attentional performance and slower reaction times, while acute exposure also disrupted neurotransmitter networks but showed less pervasive long-term alterations in dopamine signaling [163]. Similarly, in hippocampal and cortical circuits, chronic irradiation produced thinning of postsynaptic densities, changes in axon myelination, and enduring impairments in synaptic plasticity, changes that acute exposures appeared to trigger less robustly or more transiently [162]. Even in open-field navigation tasks, acute irradiation caused more pronounced, immediate deficits in spatial navigation under lighted conditions, whereas chronic dosing led to subtler but longer-lasting alterations in path integration and exploration [164]. The comparison extends to mixed-field studies where fractionated or prolonged exposures impaired novel object recognition and fear conditioning more consistently than single acute doses [171]. These investigations suggest that chronic regimens do not simply deliver a diluted version of acute effects over time but instead engage biological processes that cumulatively reshape neural networks, behavior, and cancer risk profiles in ways that are especially relevant to the continuous irradiation astronauts would experience on extended missions.

A special entry in the data set, the NASA Twins Study provides an integrative framework to understand how long-duration spaceflight affects human biology across molecular, cellular, and cognitive domains. Rather than acting as discrete stressors, galactic cosmic radiation (GCR), microgravity, circadian disruption, and confinement appear to converge on shared pathways regulating genomic stability, immune resilience, and normal neurocognitive function. Notably, chronic exposure to GCR was shown to define genome instability in this mission, evidenced by the accumulation of chromosomal inversions and other cytogenetic aberrations that persisted for months after return. The observed telomere elongation during flight, followed by pronounced shortening upon landing, raises critical questions about whether this dynamic reflects transient adaptive remodeling or persistent genomic vulnerability. Similarly, the DNA damage responses triggered by sustained GCR exposure may remain incompletely resolved, potentially elevating oncogenic risk over time. Beyond genomic instability, the study implicates coordinated shifts in transcriptional networks and microbiome composition that may modulate inflammatory tone and metabolic regulation. The persistence of gene expression perturbations, along with sustained alterations in immune mediators and cognitive performance, suggests that protective measures during spaceflight remain insufficient. These observations suggest that the interplay of GCR and other spaceflight stressors drives a complex, multi-system remodeling whose health consequences likely extend well beyond the mission timeframe [175].

Such findings reflect a pivotal turning point in space radiation biology, enabled in large part by NASA's strategic investment in GCRsim technology and the development of multibeam irradiation platforms like those at the NASA Space Radiation Laboratory (NSRL). The ability to deliver rapid, sequential exposures of biologically relevant ions—mirroring real cosmic ray compositions—has significantly advanced the field from single-ion approximations to nuanced, systems-level modeling. Coupled with omics-driven analyses, behavioral assays, and translational studies like the NASA Twins Study, this technological leap allows for integrative frameworks that capture the interactive, nonlinear nature of spaceflight stressors. As long-duration missions to the Moon and Mars approach, future research will increasingly depend on refining these models, identifying individual susceptibility markers, and testing personalized or adaptive countermeasures that address the cumulative burden of mixed-radiation and environmental exposures.

While the spaceflight context has catalyzed some of the most advanced modeling of mixed radiation effects, such scenarios are not directly representative of terrestrial conditions. Nevertheless, the principles uncovered—such as synergistic interactions, timing-dependent outcomes, and system-level disruption—stress the broader need to investigate combined exposures on Earth. Although everyday environments do not involve showers of galactic ions, people are routinely subjected to overlapping or sequential low-LET sources, radionuclide mixtures, and co-exposures with chemical, thermal, or biological stressors. The insights from GCRsim studies thus can serve as a scientific impetus, urging environmental radiation research to move beyond isolated-dose models and account for real-world exposure complexity.

On Earth, mixed radiation exposures can arise in various contexts, such as ultraviolet radiation in combination with background gamma fields in high natural radiation areas, or beta–gamma emissions associated with radionuclide contamination from industrial or military activity. Although these exposures differ in scale and composition from those encountered in space or laboratory models, they may still present layered biological challenges. As evidence suggests that interactions between radiation types can diverge from simple additive expectations, closer examination is in order. Continued investigation into such terrestrial scenarios is important for refining our understanding of mixed exposure effects, supporting environmental protection strategies, and strengthening risk assessment and remediation frameworks.

4.5. Methodological Challenges and Experimental Gaps

This review identifies major gaps in combinatorial radiation research. Some radiation pairings—such as protons with UV or beta ([16,17,18,170])—remain largely underexplored despite potential biological significance. Even for more extensively studied combinations like X-ray – neutron or X-ray – alpha, systematic mapping of dose-effect relationships and mechanistic modeling is often lacking.

Moreover, although timing, dose ratio, and radiation order have emerged as critical parameters modulating synergy, many studies treat these variables as secondary rather than primary design features. Systematic investigations of inter-exposure intervals, particularly in time-resolved or cell cycle–synchronized systems, are relatively rare and represent an important area for future study. Cross-comparative work that spans cell type, species, or tissue specificity is also scarce, making it difficult to generalize findings across biological contexts.

Another under-addressed area involves combinatorial exposures including more than two radiation types. While many studies have tested complex mixtures such as 5- or 33-ion beams in space radiation models, terrestrial analogs involving triplet exposures (e.g., UV – gamma – alpha or beta) have received little attention to date. There is also a clear need for more integrative studies that combine ionizing and non-ionizing modalities in realistic exposure sequences, such as low-dose UV exposure followed by diagnostic imaging or therapeutic irradiation.

Furthermore, many existing studies have predominantly focused on acute, high-dose exposures, whereas real-world scenarios—whether medical, environmental, or space-related— involve chronic or fractionated mixed exposures. Therefore, experimental paradigms that more closely reflect the complexity of human exposure patterns are required. Bridging these gaps will be important for constructing predictive models of mixed-radiation effects, optimizing therapeutic combinations, and refining risk estimates for spaceflight and environmental contamination scenarios. Achieving this will require interdisciplinary collaboration and coordinated efforts to standardize experimental protocols. Fortunately, recent advances in dosimetry, high-throughput molecular assays, and computational modeling now provide the necessary tools to systematically dissect mixed-radiation effects across diverse biological systems and exposure conditions. Effectively leveraging these capabilities is critical for the field to move beyond partial or descriptive observations and toward more comprehensive mechanistic understanding and improved predictive accuracy.

In addition to possible limitations in the available studies, this systematic review presented several methodological challenges of its own. To begin with, no formal review protocol was prepared and registered, and therefore other research teams cannot fully reconstruct the initial scope or trace every decision made throughout the review process. Moreover, as previously discussed, the diversity of biological systems, endpoints, and study objectives limited the relevance of conventional appraisal tools. Consequently, narrative synthesis was selected over statistical approaches due to wide data variability, which made quantitative analysis unfeasible. While appropriate for such data, a potential drawback is that this method relied more on expert judgment to interpret and integrate findings across studies. Furthermore, the search process, while broad and iteratively refined across multiple databases, was conducted manually, introducing a minor risk of omission despite multiple rounds of reviewer validation.

Another methodological concern arose from the wide temporal distribution of studies across radiation combinations. Certain pairings—notably UV and X-rays—were predominantly investigated during the 1960s–1990s, using experimental approaches that often predate contemporary molecular techniques or the use of in vivo models. Few recent studies have revisited these combinations using modern methodologies, and therefore their effects cannot always be evaluated under current biological standards. As such, while these studies provide useful mechanistic insights, they may not fully reflect current experimental standards or align with our modern understanding of the biological complexity involved. Nevertheless, they were intentionally included to capture the full conceptual scope of mixed-radiation effects. Rather than excluding them due to age, we chose to integrate their findings carefully, using them to illuminate conserved response patterns, while avoiding unwarranted generalizations. This approach aims to preserve the scientific value of legacy data without irreparably compromising the interpretive rigor of the review.

As a closing remark, the challenges encountered in preparing this extensive review are acknowledged in the interest of methodological clarity, yet they do not compromise the coherence or scientific validity of the review’s findings.

4.6. Limitations and Future Directions

One of the main challenges in mixed radiation research lies not only in the scarcity of data, but also in how current studies are designed, conducted, and contextualized. Investigations into radiobiological, medical, and space-related exposures are often pursued within separate research domains, each shaped by its own priorities, methodologies, and regulatory frameworks. This disciplinary isolation makes it difficult to compare results, integrate findings, or construct unified frameworks for understanding biological response. As a result, efforts to build predictive models—whether theoretical, computational, or mechanistic—face significant hurdles. Such models are essential not only for estimating health risks, but also for developing effective radioprotection strategies. Yet their development is hindered by fragmented data, inconsistent endpoints, and a lack of shared experimental standards. In addition, potential synergistic effects may be shaped not only by radiation type and timing, but also by differences in dose rates among combined exposures—an underexplored variable that may significantly influence biological outcomes and remains difficult to replicate experimentally. Compounding this, most studies prioritize acute or short-term outcomes, with far fewer addressing how mixed radiation effects evolve over time, across generations, or within realistic ecological and clinical contexts. For example, the extremely low dose rates characteristic of ambient space radiation—where synergistic effects may unfold gradually over time—remain experimentally inaccessible at ground-based facilities, which reflects a fundamental limitation in our ability to model the biological complexity of real spaceflight conditions.

To move the field forward, we need better coordination between disciplines, along with shared databases that connect radiation types, biological systems, and outcomes. Examples of such efforts include the NASA Open Science Data Repository and the European Radiobiological Archives (ERA), hosted by the German Federal Office for Radiation Protection (BfS), which offer structured datasets from both spaceflight and ground-based experiments. These resources support broader comparisons and enable the identification of recurring patterns across studies. Advancing the field also demands robust international coordination, standardized methodologies, and full integration of simulation and modeling platforms into experimental design. Collectively, these steps can help unify fragmented research into a coherent framework for guiding risk assessment, medical planning, and spaceflight readiness.

5. Conclusions

This systematic review—drawing on over 170 studies and encompassing an exceptional variety of radiation pairings—demonstrates that the biological effects of combined radiation exposures are neither trivial nor reliably additive. From fundamental radiobiology to medical applications and spaceflight scenarios, evidence consistently shows that interactions between different radiation types can yield emergent effects, including both synergistic and antagonistic responses. These outcomes are often shaped by radiation quality, dose ratio, sequence, and the biological system in question.

Crucially, the findings challenge the assumption that total dose alone can predict biological risk. Instead, they point to the need for a better understanding of how different radiation types interact over time and across levels of biological organization—from molecular repair mechanisms to whole-organism physiology. Although the field remains fragmented and methodologically uneven, the available data support the development of integrative models and coordinated research strategies that more accurately reflect the complexity of mixed radiation exposures.

Conclusively, the evidence presented in this review reflects a pressing need to rethink how we approach radiation exposure in both research and practice. Whether in medical, space, or environmental contexts, the biological impact of radiation cannot be fully understood without accounting for interactions across type, order, timing, and biological scale. This complexity is not merely theoretical—it is central to designing safer therapies, preparing for deep space missions, and protecting life on Earth. Moving beyond reductionist models is essential to predict, mitigate, and ultimately manage the multifaceted nature of mixed radiation effects.