Radiomitigation by Melatonin in C57BL/6 Mice: Possible Implications as Adjuvant in Radiotherapy and Chemotherapy

Abstract

Background/aim: Melatonin (N-acetyl-5-methoxytryptamine), a chief secretory molecule of the pineal gland, has multiple properties, and numerous clinical investigations regarding its actions are in progress. This study investigated the radiomitigative role of melatonin in C57BL/6 mice.

Materials and methods: Melatonin (100 mg/kg) was orally administered once daily starting at 1 h on day 1 and subsequently every 24 h until day 7 after whole-body irradiation (WBI) and survival was monitored for 30 days. The bone marrow, spleen, and intestine were studied to evaluate the mitigative potential of melatonin after radiation-induced damage.

Results: Melatonin significantly improved the survival upto 60% and 90% after 9 Gy (lethal) and 7.5 Gy (sub-lethal) WBI, respectively. Melatonin alleviated WBI-induced myelosuppression and pancytopenia, and increased white blood cell, red blood cell, platelet, and lymphocyte (CD4⁺ and CD8⁺) counts in peripheral blood. Bone marrow and spleen cellularity were restored through enhanced haematopoiesis. Melatonin ameliorated the damage in the small intestine, and promoted recovery of villi length, crypts number, and goblet cell count.

Conclusion: Melatonin mitigates the radiation-induced injury in the gastrointestinal and haematopoietic systems. The observed radiomitigative properties of melatonin can also be useful in the context of adjuvant therapy for cancer and radiotherapy.

Keywords: Melatonin; bone marrow; cancer treatment; gastrointestinal; haematopoietic; radiomitigator.

Figure 1. Mitigative effect of orally administered single dose of melatonin after WBI (whole-body irradiation) in mice. (A) The experimental design and single dosing schemes used for the survival study. (B) Single dose of melatonin (100 mg/kg) after 1 h and 24 h of day 1 after lethal (9 Gy) irradiation is represented in the Kaplan-Meier survival curves. For survival studies, n=20 mice. (C) The Table summarises the findings of MantelCox (log-rank) and Gehan-Breslow-Wilcoxon test statistics for comparing survival responses between WBI and WBI+Melatonin at 30 days postirradiation. *p<0.05; **p<0.01; ***p<0.001.

Figure 2. Mitigative effect of multiple orally administered doses of melatonin after WBI (whole-body irradiation) in mice. (A)The experimental design and multiple dosing schemes used for the survival study. (B) Multiple melatonin doses (100 mg/kg) starting at 1 h on day 1 and then daily up to day 7 after lethal (9 Gy) irradiation is represented in the Kaplan-Meier survival curves. (C) Sub-lethal (7.5 Gy) of WBI. For survival studies, n=20 mice. (D) The Table summarises the findings of Mantel-Cox (log-rank) and Gehan-Breslow-Wilcoxon test statistics for comparing survival responses between WBI and WBI+Melatonin at 30 days post-irradiation. *p<0.05; **p<0.01; ***p<0.001.

Figure 3. Melatonin mitigates radiation-induced pancytopenia in mice. Alterations in the haematological indices in the peripheral blood by melatonin. Blood counts showing (A) WBC. (B) Haemoglobin. (C) Platelet. (D) Haematocrit and (E) RBC on days 1, 3, 7, 14, 21, and 30 post-WBI (n=5). All error bars indicate SD *p<0.05; **p<0.01; ***p<0.001.

Figure 4. Role of melatonin on functional T-cells in the peripheral blood. Representative scatter plot shows the CD8 and CD4 percentages in peripheral blood lymphocytes on the 3rd and 30th day after irradiation. (A) Control. (B) Melatonin alone. (C) 7.5 Gy. (D) 7.5 Gy+melatonin. (E) Percentage of CD4⁺CD8^– and CD4^–CD8⁺ on day 3. (F) Percentages on day 30. The data are expressed as a mean and SD (n=5).

Figure 5. Melatonin mitigates radiation-induced bone marrow failure in mice. Morphological alteration in the femur bone marrow of mice posttreated with 7 doses of melatonin: (A) Control. (B) Melatonin. (C) 7.5 Gy. (D) 7.5 Gy+Mel treatment 30 days after irradiation with 7.5 Gy. The images of H&E staining were from epiphyseal ends, 20× magnification. (D) Radiation-induced vast reduction in haematopoietic cellularity and replacement by adipocytes in bone marrow (shown by black arrow). However, (E) a significantly improved number of megakaryocytes was detected under microscopy (200× shown by the red arrow). (F) Significantly decreased numbers of adipocytes were found per field under microscopy (50×) in bone marrows of 7.5 Gy+Melatonin mice compared to 7.5 Gy group 30 days after irradiation. Data are expressed as mean±SD (n=3). *p<0.05; **p<0.01; ***p<0.001.

Figure 6. Melatonin induces recovery in the spleen after 7.5 Gy of WBI (whole-body irradiation). Representative H&E-stained section of the spleen demonstrates: (A) Normal architecture of the control spleen. Red pulp (RP) and white pulp (WP, large black dotted circle) with the T-cell zone (small black dotted circle, section. I) are separated; marginal zone and trabeculae (red arrowhead) are clearly observed. (B) The appearance of the sinusoid (red arrow) in the melatonin alone group. (C) On day 30 post-irradiation of the spleen with 7.5 Gy, WP and RP appeared disorganised and some spleen cells were vacuolated (White arrow, Section II), the giant type of the activated macrophages (black arrow) is present and haemosiderin pigments are observed (white arrowhead). (D) Administration of melatonin after radiation regenerates a colony of the megakaryocytic cells (black arrowhead) with the formation of margins of WP and RP. Section I shows detail at 100× and section II at higher magnification at 400×. n=3 mice/group.

Figure 7. Melatonin decreases mouse spleen capsule thickness after WBI (whole-body irradiation). Representative H&E-stained sections of the spleen illustrates (A) Control spleen capsule with normal thickness. (B) Spleen in the melatonin alone group. (C) Infected spleen capsule on day 30 after irradiation. (D) Melatonin treatment after 1 h of WBI appears thicker than the infected spleen and decreased space between the splenocytes is observed. (E) Quantification of change in spleen capsule thickness. (F) Representative image of the spleen on day 30 after radiation exposure. The image at higher magnification with a 50 μm scale bar. Data are expressed as mean±SD (n=3). *p<0.05; ***p<0.001.

Figure 8. Effects of melatonin on small intestine radiation-induced damage. Representative H&E-stained sections of the small intestine illustrates (A) Normal ileum with intact epithelium and short, finger-like villi. (B) Melatonin-treated alone group showing no changes in the structure of the small intestine. (C) WBI (7.5 Gy) indicating pathological changes in the structure by progressive broadening and blunting of the villi up to a loss of villi structure. (D) WBI+Melatonin treatment regenerates the crypt and villi structure. (E) Villi length. (F) Crypt width. (G) The mean crypt numbers. The right panel shows detail at higher 400× magnification, and the left panel shows at 100×. Data are expressed as mean±SD, (n=3). *p<0.05; **p<0.01; ***p<0.001.

Figure 9. Melatonin mitigates goblet cell recovery following WBI (whole-body irradiation)-induced intestinal damage in mice. Representative PASstained section of the small intestine illustrates (A) Control, (B) Melatonin alone group. (C) Reduction in goblet cells following WBI (7.5 Gy). (D) Increase in the number of goblet cells following WBI+Melatonin treatment. (E) Quantification of the goblet cell number. Values shown are the number of goblet cells per villi. Data are expressed as mean±SD (n=3). ***p<0.001.

Figure 10. Schematic representation of melatonin-mediated effects on radio sensitive organs as a radiomitigator. Radiation exposure to mice leads to the death due to gastrointestinal tract and bone marrow death, which manifested as infection and impaired absorption in radiosensitive organs. Oral administration of melatonin after the 1 h of radiation exposure in mice enhances the recovery of the hematopoietic cells in the bone marrow resulting by promoting hematopoiesis in the bone marrow and increasing the differentiation of progenitor cells, which directly alleviate the blood cells for the defence. Hematopoietic progenitor cells (HPCs) migrate into the secondary lymphoid organ such as the spleen and differentiate into megakaryocytes known as extramedullary hematopoiesisnd supply leukocytes into circulation. Melatonin enhances the recovery of crypt–villus structures, which balance the food and water uptake for the survival. Melatonin also increases the proliferation of crypt cells, which regenerate the epithelial cell in the intestine.