Long-term melatonin administration attenuates low-LET gamma-radiation-induced lymphatic tissue injury during the reproductively active and inactive phases of Indian palm squirrels (Funambulus pennanti)

Abstract

A comparative analysis of low linear energy transfer (LET) gamma-radiation-induced damage in the lymphatic tissue of a tropical seasonal breeder, Indian palm squirrel (Funambulus pennanti), during its reproductively active phase (RAP) and inactive phase (RIP) was performed with simultaneous investigation of the effects of long-term melatonin pre-treatment (100 microg/100 g body weight). A total of 120 squirrels (60 during RAP and 60 during RIP) were divided into 12 groups and sacrificed at 4, 24, 48, 72 and 168 h following 5 Gy gamma-radiation exposure; control groups were excluded from exposure. Total leukocyte count and absolute lymphocyte count (ALC) and melatonin only of peripheral blood, stimulation index, thiobarbituric-acid-reactive substances (TBARS) level, superoxide dismutase (SOD) activity, and the apoptotic index of spleen as analysed by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) noted at observed time-points were significantly reduced in melatonin pre-treated groups during RAP and RIP. Long-term melatonin pre-treatment mitigated radiation-induced alterations more prominently during RIP, as assessed by ALC, TBARS, SOD, TUNEL and caspase-3 activity, at some time-points. Our results demonstrate an inhibitory role of melatonin on caspase-3 activity in splenocytes during RAP and RIP following gamma-radiation-induced caspase-mediated apoptosis. Hence, we propose that melatonin might preserve the viability of immune cells of a seasonal breeder against background radiation, which is constantly present in the environment.

Figure 1

Effect of melatonin pre-treatment (100 μg/100 g body weight) on total leukocyte count (TLC) of Funambulus pennanti following exposure to 5 Gy γ-radiation. Animals were sacrificed after 4, 24, 48, 72 and 168 h during (a) the reproductively active phase (RAP, May) and (b) the reproductively inactive phase (RIP, December). (c) A comparison of TLC between RAP and RIP in irradiated (I) and melatonin-treated plus irradiated (MI) groups, in which controls of both phases were assumed to be 100%, was completed. Vertical bars represent mean±SEM (standard error of the mean), n _ 5 for each group. The statistical significance is indicated: a _ p<0.05 and b _ p<0.01 when compared with the control group; c _ p<0.05 and d _ p<0.01 when compared with the irradiated group. Con, control; Mel, melatonin only; Irrd, irradiation only; Mel+Irrd, melatonin treatment and irradiation.

Figure 2

Effect of melatonin pre-treatment (100 μg/100 g body weight) on absolute lymphocyte count (ALC) of Funambulus pennanti following exposure to 5 Gy γ-radiation. Animals were sacrificed after 4, 24, 48, 72 and 168 h during (a) the reproductively active phase (RAP) and (b) the reproductively inactive phase (RIP). (c) A comparison of ALC between RAP and RIP irradiated (I) and RAP and RIP melatonin-treated plus irradiated (MI) groups, in which controls of both phases were assumed to be 100%. Vertical bars represent the mean±SEM (standard error of the mean), n _ 5 for each group. The statistical significance is indicated: a _ p<0.05 and b _ p<0.01 when compared with the control group; c _ p<0.05 and d _ p<0.01 when compared with the irradiated group. Con, control; Mel, melatonin only; Irrd, irradiation only; Mel+Irrd, melatonin treatment and irradiation.

Figure 3

Stimulation index (SI) of splenocytes against concanavalin A mitogenic challenge of Funambulus pennanti following exposure to 5 Gy γ-radiation. Animals were sacrificed after 4, 24, 48, 72 and 168 h during (a) the reproductively active phase (RAP) and (b) the reproductively inactive phase (RIP). (c) A comparison of SI between RAP and RIP irradiated (I) and RAP and RIP melatonin-treated plus irradiated (MI) groups, in which controls of both phases were assumed to be 100%. Vertical bars represent mean±SEM (standard error of the mean), n _ 5 for each group. The statistical significance is indicated: b _ p<0.01 when compared with the control group; c _ p<0.05 and d _ p<0.01 when compared with the irradiated group. Con, control; Mel, melatonin only; Irrd, irradiation only; Mel+Irrd, melatonin treatment and irradiation.

Figure 4

Effect of melatonin pre-treatment (100 μg/100 g body weight) on thiobarbituric acid-reactive substances (TBARS) level in the spleen of Funambulus pennanti following exposure to 5 Gy γ-radiation. Animals were sacrificed after 4, 24, 48, 72 and 168 h during (a) the reproductively active phase (RAP) and (b) the reproductively inactive phase (RIP). (c) A comparison of the TBARS level between RAP and RIP irradiated (I) and RAP and RIP melatonin-treated plus irradiated (MI) groups, in which controls of both phases were assumed to be 100%. Vertical bars represent mean±SEM (standard error of the mean), n _ 5 for each group. The statistical significance is indicated: a _ p<0.05 and b _ p<0.01 when compared with the control group; d _ p<0.01 when compared with the irradiated group only. Con, control; Mel, melatonin only; Irrd, irradiation only; Mel+Irrd, melatonin treatment and irradiation.

Figure 5

Effect of melatonin pre-treatment (100 μg/100 g body weight) on superoxide dismutase (SOD) activity of the spleen of Funambulus pennanti following exposure to 5 Gy γ-radiation. Animals were sacrificed after 4, 24, 48, 72 and 168 h during (a) the reproductively active phase (RAP) and (b) the reproductively inactive phase (RIP). (c) A comparison of SOD activity between RAP and RIP irradiated (I) and RAP and RIP melatonin-treated plus irradiated (MI) groups, in which controls of both phases were assumed to be 100%. Vertical bars represent mean±SEM (standard error of the mean), n _ 5 for each group. The statistical significance is indicated: a _ p<0.05 and b _ p<0.01 when compared with the control group; c _ p<0.05 and d _ p<0.01 when compared with the irradiated group. Con, control; Mel, melatonin only; Irrd, irradiation only; Mel+Irrd, melatonin treatment and irradiation.

Figure 6

DNA fragmentation detected using an apoptosis detection TUNEL kit. TUNEL-negative staining in (a) the control group and (b) the melatonin-treated group. (c) TUNEL-positive staining in the γ-irradiated group. (d) Melatonin decreased the number of TUNEL-positive cells. Cells visualised at 400× magnification: cells indicated with orange arrows are apoptotic and those indicated with black arrows are non-apoptotic.

Figure 7

Inhibitory effect of melatonin pre-treatment on apoptosis or TUNEL positivity index of splenocytes of Funambulus pennanti following exposure to 5 Gy γ-radiation. Animals were sacrificed after 4, 24, 48, 72 and 168 h during (a) the reproductively active phase (RAP) and (b) the reproductively inactive phase (RIP). (c) A comparison of the TUNEL positivity index between RAP and RIP irradiated (I) and RAP and RIP melatonin-treated plus irradiated (MI) groups, in which controls of both phases were assumed to be 100%. Vertical bars represent mean±SEM (standard error of the mean), n _ 5 for each group. The statistical significance is indicated: b _ p<0.01 when compared with the control group; d _ p<0.01 when compared with the irradiated group. Con, control; Mel, melatonin only; Irrd, irradiation only; Mel+Irrd, melatonin treatment and irradiation.

Figure 8

Effect of melatonin on 5 Gy γ-radiation-induced caspase-3 activity in the splenocytes of Funambulus pennanti. Animals were sacrificed after 4 h exposure during (a) the reproductively active phase (RAP) and (b) the reproductively inactive phase (RIP). (c) A comparison of caspase-3 activity between RAP and RIP irradiated (I) and RAP and RIP melatonin-treated plus irradiated (MI) groups, in which controls of both phases were assumed to be 100%. Vertical bars represent mean±SEM (standard error of the mean), n _ 5 for each group. The statistical significance is indicated: a _ p<0.05, b _ p<0.01 when compared with the control group; d _ p<0.01 when compared with the irradiated group. Con, control; Mel, melatonin only; Irrd, irradiation only; Mel+Irrd, melatonin treatment and irradiation.