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Although the association between radiation exposure and thyroid carcinogenesis is epidemiologically evident, ’true’ radiation-induced cancers cannot be identified from biological evidence of radiation-associated cases. To assess the individual risk for thyroid cancer due to radiation exposure, we aimed to identify biomarkers that are specifically altered during thyroid carcinogenesis after irradiation in a time-dependent manner in an animal model. Thyroid glands were obtained from rats (n = 175) at 6–16 months after local X-ray (0.1–4 Gy) irradiation of the neck at 7 weeks of age. The gene expression profile in thyroid glands was comprehensively analyzed using RNA microarray. Subsequently, the expression levels of the genes of interest were verified using droplet digital PCR (ddPCR). The expression level of candidate genes as biomarkers for irradiated thyroid was examined in a randomized, controlled, double-blind validation study (n = 19) using ddPCR. The incidence of thyroid cancer increased in a dose- and time-dependent manner and was 33% at 16 months after irradiation with 4 Gy. The Ki-67 labeling index in non-tumorous thyroid was significantly higher in the exposed group than in the control. Comprehensive analysis identified radiation-dependent alteration in 3329 genes. Among them, ddPCR revealed a stepwise increase in CDKN1A expression from early pre-cancerous phase in irradiated thyroid compared to that in the control. The irradiated thyroids were accurately distinguished (positive predictive value 100%, negative predictive value 69%) using 11.69 as the cut-off value for CDKN1A/β-actin. Thus, CDKN1A expression can be used as a biomarker for irradiated thyroid glands at the pre-cancerous phase.

Childhood radiation exposure is a known thyroid cancer risk factor. This study evaluated the effects of age on radiation-induced thyroid carcinogenesis in rats irradiated with 8 Gy X-rays. We analyzed cell proliferation, cell death, DNA damage response, and autophagy-related markers in 4-week-old (4W) and 7-month-old (7M) rats and the incidence of thyroid tumors in 4W, 4-month-old (4M), and 7M rats 18 months after irradiation. Cell death and DNA damage response were increased in 4W rats compared to those in controls at 1 month post-irradiation. More Ki-67-positive cells were observed in 4W rats at 12 months post-irradiation. Thyroid tumors were confirmed in 61.9% (13/21), 63.6% (7/11), and 33.3% (2/6) of irradiated 4W, 4M, and 7M rats, respectively, compared to 0%, 14.3% (1/7), and 16.7% (1/6) in the respective nonirradiated controls. There were 29, 9, and 2 tumors in irradiated 4W, 4M, and 7M rats, respectively. The expression of several autophagy components was downregulated in the area surrounding radiation-induced thyroid carcinomas in 4W and 7M rats. LC3 and p62 expression levels decreased in radiation-induced follicular carcinoma in 4W rats. Radiosensitive cells causing thyroid tumors may be more prevalent in young rats, and abrogation of autophagy may be associated with radiation-induced thyroid carcinogenesis., Scientific Reports, 11, art. no. 19096; 2021

Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient’s own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3?h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture., Scientific Reports, 7, 8447; 2017

Exposure to ionized radiation in childhood has been recognized as a risk factor for the development of thyroid cancer and possibly for other thyroid disorders. However, the effects of neonatal radiation exposure on thyroid morphology and functions have never been explored despite its potential importance. One-week-old male Wistar rats were subjected to cervical X-irradiation at 6 and 12 Gy. Animals were examined at the ages of 2, 8 and 18 weeks old. For comparison, 8-week-old rats were cervically X-irradiated at the same doses. Thyroid histology was examined by computer-assisted microscopy to measure areas of colloid and epithelium of thyroid follicles as well as epithelial heights. In rats that received cervical X-irradiation at 1 week old, the colloid size of thyroid follicles decreased at the age of 8 weeks old in a radiation-dose dependent manner. This morphological change was persistently found at 18 weeks old. There were no significant differences in serum total T3 or T4 levels among the groups. Serum TSH levels increased significantly in 8-week-old rats neonatally X-irradiated. Thyroglobulin (Tg) mRNA and protein expressions were significantly decreased in the neonatally-irradiated group while thyroid peroxidase mRNA express increased at 18 weeks old. None of these changes were observed in the rats X-irradiated at 8 weeks old. In conclusion, our results clearly demonstrated that neonatal rat thyroid was sensitive to ionized radiation, developing specific morphological changes characterized by smaller thyroid follicles along with changes in serum TSH levels and Tg expressions in the thyroid tissue.

This study aims to examine the effects of cystine and theanine (CT), which increases glutathione biosynthesis, on the survival rate and acute radiation injury of the small intestine and bone marrow using a rat model. CT pre-treatment (280 mg/kg for 5 days) significantly improved weight loss and survival rate of rats as compared with the control group after 5 Gy. CT pre-treatment significantly increased the rate of mucosa and crypt length, and decreased the number of apoptotic cells, TUNEL and cleaved caspase-3 positive cells, while increasing the number of mitotic cells and Ki-67 positive cells in jejunal crypts and villi compared to control rats post-irradiation. CT also suppressed bone marrow cell loss and reduced the number of apoptotic cells in bone marrow. These results suggest a protective effect of CT pre-treatment for acute injury after irradiation through apoptosis inhibition and increased proliferative activity in jejunal crypt cells and bone marrow cells., Environmental Toxicology and Pharmacology, 78, art.no.103395; 2020

Exposure to ionizing radiation during childhood is a well-known risk factor for thyroid cancer. Our study evaluated the effect of age on the radiosensitivity of rat thyroid glands. Four-week-old (4W), 7 -week-old (7W), and 8-month-old (8M) male Wistar rats were exposed to 8 Gy of whole-body X-ray irradiation. Thyroids were removed 3-72 h after irradiation, and non-irradiated thyroids served as controls. Ki67-positivity and p53 binding protein 1 (53BP1) focus formation (a DNA damage response) were evaluated via immunohistochemistry. Amounts of proteins involved in DNA damage response (p53, p53 phosphorylated at serine 15, p21), apoptosis (cleaved caspase-3), and autophagy (LC3, p62) were determined via western blotting. mRNA levels of 84 key autophagy-related genes were quantified using polymerase chain reaction arrays. Ki67-positive cells in 4W (with high proliferative activity) and 7W thyroids significantly decreased in number post-irradiation. The number of 53BP1 foci and amount of p53 phosphorylated at serine 15 increased 3 h after irradiation, regardless of age. No increase in apoptosis or in the levels of p53, p21 or cleaved caspase-3 was detected for any ages. Levels of LC3-II and p62 increased in irradiated 4W but not 8M thyroids, whereas expression of several autophagy-related genes was higher in 4W than 8M irradiated thyroids. Irradiation increased the expression of genes encoding pro-apoptotic proteins in both 4W and 8M thyroids. In summary, no apoptosis or p53 accumulation was noted, despite the expression of some pro-apoptotic genes in immature and adult thyroids. Irradiation induced autophagy in immature, but not in adult, rat thyroids., Journal of Radiation Research, 56(3), pp.493-501; 2015

A high dose of ionizing external radiation damage to the skin and soft tissue results in changes in function as well as in the general body condition. Once radiation surpasses the tissue safety or survival level, progressive alteration in the damaged tissue results in tissue loss and then flap loss. Local expression and action of stem cells or local growth factors in the irradiated tissue is mitigated, and external administration is sought to investigate the possibility of skin and soft tissue survival after an elevating flap. Basic fibroblast growth factor (bFGF) is primarily considered as a potent angiogenic growth factor. In burns, resurfacing with a dermal component is required, and bFGF stimulates wound healing and enhances human skin-derived mesenchymal stem cells under serum-free conditions in a dose-dependent manner. Thirty-five male, 4- to 8-week-old CLAWN miniature pigs received radiation exposure to assess the effectiveness of bFGF in terms of the progressive clinical course relevant to human skin and soft tissue. At 2 weeks following 10-Gy irradiation, tissue was preserved in the group receiving subcutaneous placement of a round-type tissue expander and bFGF. The expander plus bFGF group demonstrated significantly greater dermo-epidermal proliferation than the radiation alone, radiation plus bFGF, or expander plus radiation plus vehicle-solution groups, and new blood vessel formation was significantly increased in the expander tissue with bFGF after irradiation (p

We previously reported that the apoptosis index in jejunal crypt cells after X irradiation was greater in spontaneously hypertensive rats than in Wistar-Kyoto rats. Moreover, these same cells showed a suppression of apoptosis when reserpine was administered to induce sympathetic dysfunction in spontaneously hypertensive rats or Wistar-Kyoto rats.1) Whether the hyperfunction of the sympathetic nervous system is involved in the high susceptibility of the jejunal crypt cells to radiation-induced apoptosis was the subject of this study. The effect of norepinephrine (NE) on cell survival was examined using the colony formation assay after X-ray irradiation of rat ileal epithelial cells (IEC-18). The addition of 1 μM NE decreased the surviving fraction of cells irradiated with 6 Gy from 37% to 8%. The radiosensitivity of IEC-18 cells was enhanced by the addition of 1 μM of NE. The irradiation and treatment with NE also resulted in an increased cellular apoptotic rate. These results showing enhanced radiosensitivity of rat ileal epithelial cells by NE suggest that NE may be one of the factors which aggravate acute radiation injury in the intestine., Journal of Radiation Research, 52(3), pp.369-373; 2011

The effect of basic fibroblast growth factor (bFGF) was studied in radiation-induced apoptosis in rat jejunal crypt cells. Six-week-old male Wistar rats were administered 4 mg/kg bFGF intraperitoneally 25 h before receiving 8 Gy whole-body X rays. The jejunum was removed for analysis from time 0 to 120 h after irradiation. Villus length in control rats declined steadily until 72 h, while in bFGF-treated rats the villi were longer than in the controls until 48 h. Crypt lengths were similar to villi. bFGF treatment increased Ki-67-positive cells in the jejunal crypt at 0, 24 and 48 h. The treatment with bFGF reduced the number of apoptotic cells per jejunal crypt to 23% and 10% of the control values at 3 and 6 h, respectively, and increased numbers of mitotic cells significantly at 48 and 72 h. bFGF decreased the levels of TP53, CDKN1A, Puma and Cleaved caspase 3 at 3 h as detected by Western blot analyses. Our results suggest that bFGF protected against acute radiation-induced injury by suppressing the crypt apoptotic cells including the stem cells and promoted crypt cell proliferation. The inhibition of apoptosis thus might be related to suppression of the TP53 pathway., Radiation research, 174(1), pp.52-61; 2010

We postulated that nuclear dust within the lamina propria beneath the basement membrane of the epithelium in colonic mucosa is a form of apoptotic epithelial cells and that its expression triggers dextran sulfate sodium-induced colitis. The aim was to determine the origin of nuclear dust and to explore the correlation between nuclear dust expression and clinicopathologic parameters of colitis. Rats were treated with 3% dextran sulfate sodium. Cells showing double positive staining with cytokeratin and TdT-mediated uUTP-biotin nick-end labeling technique were apoptotic cells derived from epithelial cells. Nuclear dust expression on day 5 correlated with bloody stools and a decrease of mitotic colonic cells just before ulceration. Examination of cultures under light and fluorescent microscopy showed that dextran sulfate sodium caused early apoptosis and late apoptosis or necrosis. Our results suggest that interventions directed toward the apoptotic process may be beneficial in the treatment of ulcerative colitis.

Polaprezinc, an anti-ulcer drug, is a chelate compound consisting of zinc and L-carnosine. Polaprezinc has been shown to prevent gastric mucosal injury. The anti ulcer effects of polaprezinc have been ascribed to its antioxidative property. The effect of polaprezinc on ionizing radiation-induced apoptosis was studied in the jejunal epithelial crypt cells of rats. Seven-to eight week-old Wistar rats, which were treated with 100 mg/kg of polaprezinc orally 1h before irradiation or 2% carboxymethyl cellulose sodium in controls, were exposed to whole body X-ray irradiation at 2 Gy. The number of apoptotic cells per jejunum crypt was counted in haematoxylin and eosin stained sections at 0-6 h after irradiation. TUNEL positive cells and immunopositive cells for active caspase-3 per crypt were also counted. Accumulation of p53, p21^ and Bax expression in the jejunum after irradiation were examined by Western blot analyses. Polaprezinc treatment given prior to radiation resulted in a significant reduction in numbers of apoptotic cells, TUNEL positive cells and active caspase-3 immunopositive cells in jejunal crypt cells. Polaprezinc treatment resulted in decreases of p53 accumulation, p21^ and Bax expression after irradiation. Polaprezinc has a protective effect against ionizing radiation induced apoptosis in rat jejunal crypt cells., Journal of Radiation Research, 49(4), pp.341-347; 2008

X rays are well known to cause genetic damage and to induce many types of carcinomas in humans. The Apc(min/+) mouse, an animal model for human familial adenomatous polyposis (FAP), contains a truncating mutation in the APC gene and spontaneously develops intestinal adenomas. To elucidate the role of X rays in the development of intestinal tumors, we examined the promotion of carcinogenesis in X-irradiated Apc(min/+) mice. Forty out of 77 (52%) X-irradiated Apc(min/+) mice developed adenocarcinomas that invaded the proprial muscle layer of the small intestine; 24 of 44 (55%) were in males, and 16 of 33 (49%) were in females. In contrast, invasive carcinomas were detected in the small intestines of only 13 of 64 (20%) nonirradiated Apc(min/+) mice; nine of 32 (28%) were in males and four of 32 (13%) were in females. These differences between X-irradiated and nonirradiated Apc(min/+) mice in the occurrence of invasive intestinal carcinomas were statistically significant (P0.05 for males, P0.005 for females). In wild-type mice, invasive carcinomas were not detected in either X-irradiated or nonirradiated mice. Apc(min/+) mice had many polyps in the large intestine with or without X irradiation; there was no difference in the number of polyps between the two groups. Also, invasive carcinomas were not detected in the large intestine with or without irradiation. The occurrence of mammary tumors, which was observed in Apc(min/+) mice, was found to be increased in irradiated Apc(min/+) mice (P0.01). Apc(min/+) mice had many polyps in the small and large intestines with or without X irradiation. X-irradiated Apc(min/+) mice had highly invasive carcinomas in the small intestine with multiplicities associated with invasiveness. Our results suggest that X radiation may promote the invasive activity of intestinal tumors in Apc(min/+) mice.

Interleukin-11/PI3K/X-ray Irradiation/Intestinal Epithelial Cell. Interleukin-11 (IL-11) is a well known anti-inflammatory cytokine that is associated with cell growth, and also participates in limiting X-ray irradiation induced intestinal mucosal injury. The aim of this study was to evaluate the protective effect of IL-11 on the cell injury induced by X-ray irradiation in rat intestinal epithelial IEC-18 cells. Recombinant human IL-11 (rhIL-11) treated cells were irradiated and then examined for cell viability. To evaluate irradiation injury, trypan blue staining was used to detect the dead cells. The viability of irradiated cells was up-regulated by rhIL-11 treatment and also resulted in the activation of p90 ribosomal S6 kinase (p90RSK) and S6 ribosomal protein (S6Rp). Wortmannin, a specific inhibitor of PI3K, suppressed the activation of S6Rp in rhIL-11 treated cells, and decreased the up-regulation of viability by rhIL-11 treatment in irradiated cells. The TUNEL assay was also perfomed to estimate the rate of apoptosis in X-ray induced cell death. There was no difference in the results between trypan blue staining and the TUNEL assay. Further, rhIL-11 down-regulated the expression of cleaved caspase-3 in irradiated cells. These results suggest that rhIL-11 may play an important role in protection from radiation injury., Journal of radiation research. 2007, 48(2), p.171-177

Radiotherapy for malignant pelvic disease is often followed by acute radiation colitis (ARC). It has been reported that sucralfate treatment has a protective effect against ARC, though the mechanisms of action are unknown. The effects of sucralfate on X-ray radiation-induced apoptosis was studied at 4 Gy in the colonic crypt cells of rats. Sucralfate enemas given prior to radiation resulted in the following: (1) reduction in number of apoptotic colonic crypt cells; (2) reduction in number of caspase-3 positive cells; (3) decreases in p53 accumulation and p21 expression; (4) decreases of Bax/Bcl-2 ratio. The protective effects of sucralfate against ARC may be partially due to the suppression of radiation-induced apoptosis by way of p53 in the colon and the protection of the colonic epithelial stem cell region., Journal of radiation research. 2006, 47(1), p.1-8

A high dose of ionizing external radiation damage to the skin and soft tissue results in changes in function as well as in the general body condition. Once radiation surpasses the tissue safety or survival level, progressive alteration in the damaged tissue results in tissue loss and then flap loss. Local expression and action of stem cells or local growth factors in the irradiated tissue is mitigated, and external administration is sought to investigate the possibility of skin and soft tissue survival after an elevating flap. Basic fibroblast growth factor (bFGF) is primarily considered as a potent angiogenic growth factor. In burns, resurfacing with a dermal component is required, and bFGF stimulates wound healing and enhances human skin-derived mesenchymal stem cells under serum-free conditions in a dose-dependent manner. Thirty-five male, 4- to 8-week-old CLAWN miniature pigs received radiation exposure to assess the effectiveness of bFGF in terms of the progressive clinical course relevant to human skin and soft tissue. At 2 weeks following 10-Gy irradiation, tissue was preserved in the group receiving subcutaneous placement of a round-type tissue expander and bFGF. The expander plus bFGF group demonstrated significantly greater dermo-epidermal proliferation than the radiation alone, radiation plus bFGF, or expander plus radiation plus vehicle-solution groups, and new blood vessel formation was significantly increased in the expander tissue with bFGF after irradiation (p 0.01). Electron microscopy revealed that tissue with expander and bFGF maintained more stable skin adnexae with preserved intact epidermis and dermis. Thus, bFGF improved and maintained the tissue viability after immediate irradiation in the skin and soft tissue.

Epidermal cells are the first cells to be exposed to environmental genotoxic agents such as ultraviolet (UV) and ionizing radiations (IR), which induce DNA double strand breaks (DSBs) and activate DNA damage response (DDR) to maintain genomic integrity. Defective DDR can result in genomic instability (GIN) which is considered to be a central aspect of any carcinogenic process. P53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DSBs and rapidly form nuclear foci, the presence of 53BP1 nuclear foci can be considered as a cytological marker for endogenous DSBs reflecting GIN. The levels of GIN were analyzed by immunofluorescence studies of 53BP1 in 56 skin tumors that included 20 seborrheic keratosis (SK), 8 actinic keratosis (AK), 9 Bowen’s disease (BD), 9 squamous cell carcinoma (SCC), and 10 basal cell carcinoma (BCC). This study demonstrated a number of nuclear 53BP1 foci in human skin tumorigenesis, suggesting a constitutive activation of DDR in skin cancer cells. Because AK showed a high DDR type of 53BP1 immunoreactivity, GIN seems to be induced at the precancerous stage. Furthermore, invasive cancers exhibited a high level of intense, abnormal 53BP1 nuclear staining with nuclear accumulation of p53, suggesting a disruption of DDR leading to a high level of GIN in cancer cells. The results of this study suggest that GIN has a crucial role in the progression of skin carcinogenesis. The detection of 53BP1 expression by immunofluorescence can be a useful histological marker to estimate the malignant potential of human skin tumors., Cancer science, 99(5), pp.946-951; 2008

Defective DNA damage response (DDR) can result in genomic instability (GIN) and lead to the transformation into cancer. P53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DNA double strand breaks (DSBs) and rapidly form nuclear foci, the presence of 53BP1 foci can be considered as a cytologic marker for endogenous DSBs reflecting GIN. Although it has been proposed that GIN has a crucial role in the progression of thyroid neoplasms, the significance of GIN during thyroid tumorigenesis remains unclear, particularly in patients. We analyzed, therefore, the level of GIN, as detected with immunofluorescence of 53BP1, in 40 cases of resected thyroid tissues. This study demonstrated a number of nuclear 53BP1 foci in thyroid cancers, suggesting a constitutive activation of DDR in thyroid cancer cells. Because follicular adenoma also showed a few 53BP1 nuclear foci, GIN might be induced at a precancerous stage of thyroid tumorigenesis. Furthermore, high-grade thyroid cancers prominently exhibited an intense and heterogeneous nuclear staining of 53BP1 immunoreactivity, which was also observed in radiation-associated cancers and in mouse colonic crypts as a delayed response to a high dose ionizing radiation, suggesting increased GIN with progression of cancer. Thus, the present study demonstrated a difference in the staining pattern of 53BP1 during thyroid carcinogenesis. We propose that immunofluorescence analysis of 53BP1 expression can be a useful tool to estimate the level of GIN and, simultaneously, the malignant potency of human thyroid tumors.