Cyclin D1 staining is greater after ⁵⁶Fe radiation.

<p>A) Representative images of cyclin D1 stained tumor free (normal) areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. B) Quantification of cyclin D1 staining in tumor free areas of control, γ, and ⁵⁶Fe-irradiated sections. C) Representative images of cyclin D1 stained tumor bearing areas of intestinal sections from control, γ-, and ⁵⁶Fe-irradiated mice. D) Quantification of cyclin D1 staining in tumor bearing areas of control, γ, and ⁵⁶Fe-irradiated sections.</p

Heavy Ion Radiation Exposure Triggered Higher Intestinal Tumor Frequency and Greater β-Catenin Activation than γ Radiation in APC^Min/+ Mice

<div><p>Risk of colorectal cancer (CRC) after exposure to low linear energy transfer (low-LET) radiation such as γ-ray is highlighted by the studies in atom bomb survivors. On the contrary, CRC risk prediction after exposure to high-LET cosmic heavy ion radiation exposure is hindered due to scarcity of <i>in vivo</i> data. Therefore, intestinal tumor frequency, size, cluster, and grade were studied in APC^Min/+ mice (n = 20 per group; 6 to 8 wks old; female) 100 to 110 days after exposure to 1.6 or 4 Gy of heavy ion⁵⁶Fe radiation (energy: 1000 MeV/nucleon) and results were compared to γ radiation doses of 2 or 5 Gy, which are equitoxic to 1.6 and 4 Gy ⁵⁶Fe respectively. Due to relevance of lower doses to radiotherapy treatment fractions and space exploration, we followed 2 Gy γ and equitoxic 1.6 Gy ⁵⁶Fe for comparative analysis of intestinal epithelial cell (IEC) proliferation, differentiation, and β-catenin signaling pathway alterations between the two radiation types using immunoblot, and immunohistochemistry. Relative to controls and γ-ray, intestinal tumor frequency and grade was significantly higher after ⁵⁶Fe radiation. Additionally, tumor incidence per unit of radiation (per cGy) was also higher after ⁵⁶Fe radiation relative to γ radiation. Staining for phospho-histone H3, indicative of IEC proliferation, was more and alcian blue staining, indicative of IEC differentiation, was less in ⁵⁶Fe than γ irradiated samples. Activation of β-catenin was more in ⁵⁶Fe-irradiated tumor-free and tumor-bearing areas of the intestinal tissues. When considered along with higher levels of cyclin D1, we infer that relative to γ radiation exposure to ⁵⁶Fe radiation induced markedly reduced differentiation, and increased proliferative index in IEC resulting in increased intestinal tumors of larger size and grade due to preferentially greater activation of β-catenin and its downstream effectors.</p> </div

Antioxidant enzyme activity in IEC.

<p>A) SOD1 activity after exposure to γ and ⁵⁶Fe radiation. B) SOD2 activity after exposure to γ and ⁵⁶Fe radiation. C) Catalase activity after exposure to γ and ⁵⁶Fe radiation. *significant compared to control; **significant compared to γ radiation.</p

Heavy ion radiation-induced intestinal tumorigenesis in APC^Min/+ mice.

<p>Intestinal tumor induction after 1.6 Gy or 4 Gy of ⁵⁶Fe radiation is compared to respective equitoxic doses γ radiation (2 Gy γ equitoxic to 1.6 Gy ⁵⁶Fe and 5 Gy γ equitoxic to 4 Gy ⁵⁶Fe) as well as to sham-irradiated controls.</p

⁵⁶Fe radiation-induced larger and higher-grade intestinal tumors.

<p>A) Compared to γ radiation, exposure to ⁵⁶Fe radiation led to greater number of tumors whose size was ≥3 mm. B) H&E stained intestinal tumors showing crypt penetration of mucularis mucosa (arrow) indicating invasive adenocarcinoma after ⁵⁶Fe radiation. Tumors in control and γ irradiated mice were mostly adenomas. C) Percent of invasive adenocarcinoma in control, γ, and ⁵⁶Fe irradiated tumors.</p

Measuring ROS and mitochondrial superoxide in IEC.

<p>A) Flow cytometry histogram showing change in ROS level after exposure to γ, and ⁵⁶Fe radiation. B) Quantification of ROS level presented as percent change of mean fluorescence. C) Flow cytometry histogram showing mitochondrial superoxide levels after exposure to γ and ⁵⁶Fe radiation. D) Quantification of mitochondrial superoxide level presented as percent change of mean fluorescence. Cells from unirradiated mice were used as controls. *significant compared to control; **significant compared to γ radiation. Histogram colors - Black: control, Blue: γ radiation, and Red: ⁵⁶Fe radiation samples.</p

Higher oxidative DNA damage after ⁵⁶Fe radiation.

<p>A) Immunohistochemical staining of intestinal sections for 8-oxo-dG after exposure to γ and ⁵⁶Fe radiation. B) Quantification of 8-oxo-dG staining in intestinal sections. *significant compared to control; **significant compared to γ radiation.</p

Measurement of oxidative stress in IEC.

<p>A) Nitrate/nitrite levels were measured using Griess reagent in IEC after radiation exposure. B) Lipid peroxidation assessed in IEC using cis-parnaric acid is presented as mean fluorescent intensity. Decrease of cis-parnaric acid fluorescence is proportionate to increase in lipid peroxidation. C) NADPH oxidase activity was determined in IEC by measuring lucigenin luminescence after radiation exposure and is presented as relative light unit (RLU) per µg protein. Change in lucigenin luminescence is proportional to change in NADPH oxidase activity. *significant compared to control; **significant compared to γ radiation.</p

Greater phospho-histone H3 staining after ⁵⁶Fe radiation.

<p>A) Representative images of phospho-histone H3 stained tumor free (normal) areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. B) Quantification of phospho-histone H3 positive nuclei in tumor free areas of control, γ, and ⁵⁶Fe-irradiated sections. C) Representative images of phospho-histone H3 stained tumor bearing areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. D) Quantification of phospho-histone H3 positive nuclei in tumor bearing areas of control, γ, and ⁵⁶Fe-irradiated sections.</p

Schematic overview of high-LET radiation-induced persistent oxidative stress.

<p>A) Low-LET γ radiation is sparsely ionizing and has very few secondary tracks. B) A primary track of high-LET radiation is densely ionizing and has more secondary tracks than low-LET radiation. I: cell directly hit by a primary track, II: bystander cell, III: cell hit by secondary delta-ray tracks.</p