Orthovoltage X-Rays Exhibit Increased Efficacy Compared with γ-Rays in Preclinical Irradiation

Abstract

Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects.

Significance: Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.

Figure 1:. Radiation Beam Quality Alters 30-Day Survival in Mice Exposed to Whole-Body Irradiation.

(A) Schematic depicting the effects of radiation energy on bone marrow injury with calculated average energies of the radiation spectra, ratios of photoelectric effect to Compton scattering in compact bone and soft tissue, and LD₅₀ from each irradiation source. (B) Kaplan-Meier survival curves from experiments with whole-body irradiation of mice using orthovoltage X-rays with 1 mm Cu filtration, (C) orthovoltage X-rays with Thoraeus filtration, or (D) Cs-¹³⁷ γ-rays. (n≥10/group; Results pooled from several experiments.) (E) Mean weight loss (%) following whole body irradiation with orthovoltage X-rays with 1 mm Cu filtration, (F) orthovoltage X-rays with Thoraeus filtration, or (G) Cs-¹³⁷ γ-rays. (H) Logit analysis of 30-day survival using each radiation modality. (I) Kaplan-Meier curve comparing 7.0 Gy with each modality. 95% confidence intervals (dotted line). (J) Comparison of weight loss (%) following 7 Gy whole-body irradiation with each radiation modality. Data is represented as mean ± SEM. HVL: Half-Value Layer.

Figure 2:. Hematopoietic Injury Decreases with Increasing Radiation Energy at a Constant Dose.

Complete blood count of peripheral blood analyzed by one-way ANOVA for (A) hematocrit, (B) white blood cells, and (C) platelets 10-days after 7 Gy whole-body irradiation. Results combined from two independent experiments (n≥3/group). (D) Femoral cell counts were performed 10-days post-irradiation using an automated hemacytometer and combined from two independent experiments for analysis with one-way ANOVA. (E) H&E-stained sternal bone marrow depicting sternebrae 10-days post-irradiation representative of three independent experiments. (Scale Bar 100 μm; n≥6/group). (F) H&E-stained sternal bone marrow 10-days post-irradiation representative of three independent experiments (Scale Bar 20 μm; n≥6/group). (G) Prussian blue staining with nuclear fast red counterstain to detect iron (blue) in sternal bone marrow 10-days post-irradiation. Representative of three independent experiments (Scale Bar 25 μm; n≥6/group). (H) Hematopoietic stem and progenitor cell flow cytometry was performed on bone marrow collected from mice 10-days post-irradiation with results combined from two independent experiments analyzed by two-way ANOVA (n≥6/group). Populations are represented as the total number of cells per femur using logarithmic scale. Data is represented as mean ± SEM. P<.01 (**), P<.0001 (****). LT-HSC: Long-Term Hematopoietic Stem Cell, MPP: Multipotential Progenitor, CMP: Common Myeloid Progenitor, GMP: Granulocyte-Macrophage Progenitor, MEP: Myeloid-Erythroid Progenitor, CLP: Common Lymphoid Progenitor.

Figure 3:. Splenic and Thymic Regeneration Following 7 Gy WBI Varies with Radiation Energy.

(A) Representative images of H&E-stained thymus 10-days post-IR from two independent experiments (Scale Bar 100 μm; n≥3/group). (B) Thymus weights 10-days post-irradiation represented as a percent of body weight at the time of euthanasia and analyzed by one-way ANOVA. (C) H&E-stained spleens harvested from mice 10-days post-irradiation with regions of extramedullary hematopoiesis delineated by dotted yellow lines. Results are representative of two independent experiments. (Scale Bar 200 μm; n≥6/group). (D) Spleen weights 10-days post-IR represented as a percent of body weight at the time of euthanasia and analyzed by one-way ANOVA. (n≥3/group). (E) CD71 IHC (brown) in spleens 10-days post-irradiation, representative of two independent experiments (Scale Bar 100 μm; n≥6/group). (F) Splenic flow cytometry for the percent of live Lin-, Sca-1+, c-Kit+ (LSK) cells 10-days post-irradiation analyzed by one-way ANOVA. (n≥2/group). (G) Spleens were stained with Prussian Blue to detect iron (blue) 10-days post-irradiation. Results are representative of three independent experiments. (H) Total number of LSK cells per spleen 10-days post-irradiation analyzed by one-way ANOVA. (I) Uniform manifold approximation and projection (UMAP) plots representing clusters of immune cell populations in the spleens 10-days post-irradiation. Gating strategy at right. (J) Leukocyte populations gated on UMAP clusters as a percent of total CD45⁺ cells were compared by two-way ANOVA. Data is represented as mean ± SEM. P<.05 (*), P<.01 (**), P<.001 (***), P<.0001 (****). DN: Double Negative, NKs: Natural-Killer Cells, DCs: Dendritic Cells, pDCs: Plasmacytoid Dendritic Cells.

Figure 4:. Gastrointestinal Injury is Dependent on Energy of Irradiation

(A) Gross pathology of stomachs 10-days after whole-body irradiation demonstrating gastric hemorrhage. (B) Gross gastric bleeding was scored semi-quantitatively by a blinded observer. (C) Representative H&E-stained sections of stomachs. Yellow arrowheads indicate mucosal hemorrhage. Yellow asterisk indicates submucosal hemorrhage. (Scale Bar 50 μm; n≥4/group). (D) H&E-stained intestinal sections from mice 4-days post-12 Gy WBI representative of two independent experiments. (Scale Bar 100 μm; n≥3/group.) (E) Total counts of crypts per circumference of intestinal cross-sections and (F) Crypt depth measurements 4-days post-irradiation analyzed by one-way ANOVA. (G) EdU incorporation was fluorescently detected (green) to assess cell proliferation in intestinal crypts 4-days post-12 Gy WBI. Images representative from two independent experiments. (Scale Bar 100 μm; n≥3/group). (H) EdU⁺ cells per intestinal crypt were counted 4-days post-irradiation and analyzed by one-way ANOVA. (I) Olfactomedin-4 (OLFM4; red) was detected by IHC 4-days after 12 Gy of whole-body irradiation, representative of two independent experiments (Scale Bar 100 μm; n≥3/group). (J) The percent of regenerating crypts (≥5 EdU+ cells/crypt) was calculated 4-days post-irradiation and analyzed by one-way ANOVA. Data is represented as mean ± SEM. P<.05 (*), P<.01 (**), P<.001 (***), P<.0001 (****).

Figure 5:. Response to Partial Body-Irradiation is Altered by Energy of Irradiation

(A) Kaplan-Meier survival curves for mice receiving partial-body irradiation with either 1 mm Cu or (B) Thoraeus filtered orthovoltage X-rays. (C) Comparison of weight loss (% of starting body weight) following partial-body irradiation with either (C) 1 mm Cu or (D) Thoraeus filtration. (E) Representative Hematoxylin & Eosin (H&E)-stained intestinal sections from mice 4-days post-12 Gy partial-body irradiation. (Scale Bar 100 μm; n≥3/group.) (F) Total counts of crypts per circumference of intestinal cross-sections and (G) Crypt depth measurements 4-days post-irradiation analyzed by one-way ANOVA. (H) EdU incorporation was fluorescently detected (green) to assess cell proliferation in intestinal crypts 4-days post-12 Gy PBI (Scale Bar 100 μm; n≥3/group.) (I) EdU+ cells per intestinal crypt and (J) the percent of regenerating crypts (≥5 EdU+ cells/crypt) 4-days post-irradiation analyzed by one-way ANOVA. (K) Representative H&E-stained intestinal sections from moribund mice following 12 Gy partial-body irradiation. (Scale Bar 100 μm; n=5). (L) Representative H&E-stained bone marrow sections showing one sternebrae in moribund mice (n=5). (M) Kaplan-Meier survival curves from mice irradiated either with 12 Gy PBI (1 mm Cu) or 12 Gy WBI (Cs-¹³⁷). Mice were either administered a PBS injection or bone marrow transplant 24 hours post-irradiation. (N) Weight loss as a percent of starting body weight for mice in the survival study. Data is represented as mean ± SEM. P<.05 (*), P<.01 (**), P<.001 (***).