Infertility has been a major medical and social preoccupation. The protective ability of the phytochemicals against radiation-induced male reproductive abnormalities may offer a new insight into the modification of testicular germ-cell radiosensitivity which may have implication in amelioration of testicular injuries. Therefore, the major concern of the present investigation is to assess the possible radioprotective capability of Tinospora cordifolia extract in clinical field against radiation-induced male reproductive dysfunctions.
General observations in the present study clearly indicate that preirradiated treatment with TCE appreciably increased survival time of mice by 30 days without any symptoms of radiation-induced sickness. At the same time, irradiated animals exhibited sickness within 2–4 days with 71.43% mortality during 7 days and the remaining animals died within the next 10 days after exposure which might be occurred due to hematopoietic syndrome as suggested by others also [26
]. In view of the fact that the significant enhancement in the survival time may be owing to the protection afforded by TCE to the stem cell component of the bone marrow, which continued to supply the requisite number of cells in the survivors [28
]. Body weight, testes weight, and testes-body weight ratio exhibited a similar mode of variation in both irradiated control and TCE-treated experimental animals. A notable recovery in body weight was ensued from day 7 onward in TCE-pretreated irradiated animals but without achieving the normal weight even till the end of experiment. These results suggest the possibility of protection of gastrointestinal system by TCE, since radiation-induced loss in body weight is due to the decrease in food and water intake due to gastrointestinal damage as also described by Griffiths et al. [29
]. Some earlier studies from our laboratory also showed that administration of crude extracts of different herbal drugs reduced radiation-induced loss in body weight of animals [30
], whereas decrease in testicular weight and testicular body-weight ratio after radiation exposure may be due to the actual loss in the germinal epithelial cells and not reflected by changes in the interstitial tissue or Sertoli cells. These findings may be further supported by our experiments in which spermatogenic cells in testes were found to be greatly reduced, while interstitial and Sertoli cells appeared almost unaffected by radiation exposure (unpublished data). Similar declining pattern in testicular weights and in weight index was also observed by Lin et al. [33
] and Jagetia et al. [34
] in lethally irradiated mice.
Radiation is a potent toxicant and whole-body exposure of it can alter the general physiology of animal which might have an impulse over the normal histology and physiopathology of testes. Radiation can also downregulate its dual character, steroidogenic, and spermatogenic activity through the generation of oxidative stress, suppression of antioxidant mechanism, and by activating numerous molecular pathways involved in germ cells life and death decision making that ultimately altered normal testicular architecture [35
]. In the present experiment, histological examination of seminiferous tubules of irradiated mice showed marked pathological alterations in the form shrinkage of tubules, distortion of cellular arrangement, exfoliation, severe intertubular oedema and hemorrhage in intertubular space, karyorrhexis, karyolysis, pycnosis, and necrosis. These findings are in close agreement with the earlier report of Pareek et al. [36
] which documented the degenerative effects of gamma rays on spermatogenesis in lethally irradiated mice. The above pathological symptoms were manifested from the beginning of experimentation and increased progressively till day 15th in which germinal epithelium appeared flaccid and highly disorganized with total arrest of spermatogenesis. In addition to this, the nuclei, however, showed more destruction; they became shrunken and more concentrated with resultant karyorrhexis karyolysis and pycnosis. By the end of experiment, these nuclear and cytoplasmic changes led to necrosis and ultimately complete disappearance of the cells. Similar types of testicular injuries have also been reported by Zhang et al. [37
], Koruji et al. [38
], and Pande et al. [39
] in lethally irradiated mice.
Microscopic analysis of the testes in both irradiated control and TCE-treated animals revealed that peripheral as well as central tubular diameter of seminiferous tubules decreased progressively up to day 15 of irradiation and this might be caused by the spermatogenic cell loss and tubular disorganization. In TCE-treated irradiated animals, pathological lesions showed a similar pattern of alteration as in the irradiated control group, but their appearance was less prominent at all the autopsy intervals. TCE pretreatment rendered a high degree of recovery in spermatogenic cells, and almost a normal testicular architecture was reestablished by the end of experiment, but some pathological lesions like adhesion of tubules, mild cytoplasmic vacuolation, and less number of mature spermatozoa still persisted in the lumen of some tubules. A similar observation has also been reported while using Panax ginseng
], Podophyllum hexandrum
] and Mentha
] as radio-protector for the modulation of testicular injuries after irradiation.
The degenerative changes in testes observed at the early intervals of experiment in irradiated control may be due to immediate cell death and cellular death during their attempt to divide [43
]. Intertubular oedema and hemorrhage in Intertubular spaces are caused due to direct effect of radiation on the testes, while other pathological alterations which were observed after 1 or 2 days may be due to the effect of radiation on the other parts of the body system [44
]. Irradiated control animals showed shrunken and emptiness of seminiferous tubules that were associated with depletion in total germ cells population and breakdown of Sertoli cell-germ cell coordination links which might be responsible for the loosening and wavy appearance of tubular walls and tunica albuginea as suggested by others also [45
TCE preirradiation treatment was found to protect testicular cells very effectively which may be attributed to several factors such as efficient scavenging of free radicals, repair of DNA, membrane, and other damaged target molecules, and the replenishment of severely damaged or dead cells. The recruitment of cells to substitute the apoptatic and necrotic cells could also add to protection provided by TCE. In this study, the maximum protection is apparent after 3–7 days of radiation exposure which may be due to the fact that the optimal cellular concentration of free radical scavenging constituents of TCE is present in the system after 3–7 days.
Understanding the protective mechanism accessible by medicinal plant extracts against the consequences of repeated oxidative stress in the male reproductive milieu is gaining wide attention in the present nuclear technological environment Agarwal and Said, 2005 [47
]. Elevated levels of ROS may influence some transcription factors, enzyme activities, cell proliferation, and various important signal transduction pathways, leading to male reproductive dysfunctions (Kaur et al. [48
Keeping this view, it pertinent to assess the possible anti-oxidative role of T. cordifolia
extract against radiation-induced testicular oxidative stress. Radiation exposure induced a significant depletion in GSH levels at early intervals, which may be due to its enhanced utilization as an attempt to detoxify the acute radiation-induced free radical damage as glutathione is a major endocellular nonenzymatic antioxidant and executes its radioprotective function through free radical scavenging mechanism (Bump and Brown [49
]. Depletion of GSH was lower in TCE pretreated animals as the animals of this group had a high level of phytoantioxidants after 5 days of TCE administration, therefore, less utilization of endogenous glutathione was observed. Afterwards, it tended to be utilized less due to the declining impact of radiation and endogenous reparative homeostatic activity. Previous findings from our laboratory [50
] strongly suggest that radiation-induced depletion of glutathione resulted in an enhanced LPO as also observed in testicular tissue by Faidan et al. [52
]. Since biomembranes of testicular tissues are rich in polyunsaturated fatty acid content and radiation-induced damage is mediated by peroxidation of membrane lipids, therefore, the estimation of LPO is important to monitor oxidative damage to cellular membranes [53
LPO produces a progressive loss of cellular integrity, fluidity of sperm membrane, and its motility, impairment in membrane transport function and disruption of cellular ion homeostasis in testes (Aitken and Baker, [54
]). In the present study, both the irradiated control and experimental groups showed a gradual and continuous augmentation in the level of TBARS contents till day 15 postirradiation, which may be due to increased oxidative stress and decrease in body weight, organ weight, and protein value after radiation exposure as also suggested by Yadav et al. [55
]. The perpetuation of cellular membrane integrity depends on protection or repair mechanism capable of neutralizing oxidative reactions. Preirradiation treatment of TCE significantly reduced LPO at all the autopsy intervals in comparison to irradiated control, which testifies to our belief that one of the possible mechanisms of radioprotection by TCE may be owing to the scavenging of free radicals generated by radiation exposure and prevents the formation of endoperoxidation.
Our results suggest that TCE contains a very effective and natural antioxidant system (NAO) that is capable of preventing oxidative damage which is mediated principally through the generation of reactive oxygen species. The exact mechanism of action of TCE is not known. However, scavenging of free radicals and increased concentration of endogenous antioxidant system may be considered as important mechanisms of protection provided by TCE against radiation-induced damage to the testicular tissue. Tyagi et al. [56
] imparts the support to this contention by the experiments on free radical scavenging, where TCE has been found to scavenge radiation-mediated OH and O2−
radicals. Earlier, Goel et al. [57
] also reported the free-radical scavenging ability of aqueous extract of T. Cordifolia
. The prophylactic action of TCE against radiation-induced reproductive and metabolic disorder may be due to the presence of several bioactive constituents like glycosides [58
], phenolics [59
], alkaloids [60
], diterpenoid lactones [61
], steroids [62
], miscellaneous compounds [63
], and so forth which may act through different mechanisms such as antioxidative [64
], stimulation of cell proliferation [65
], immunomodulation [66
], and inhibition of lipid peroxidation [67
Radioprotective efficacy may be due to combined/synergistic impact of these constituents rather than to a one single factor. Many studies around the world proved that the selection of a particular food plant, plant tissue, or herb for its potential health benefits appears to mirror its polyphenol and flavonoid composition. Polyphenols act as chain breakers or radical scavengers, which attribute to their antioxidant properties possibly through their O2−.
and singlet oxygen quenching ability as also noticed by Weiss and Landauer [68
] and Hou et al. [69
]. Most polyphenols, especially flavonoids are very effective scavengers of hydroxyl and peroxyl radicals [70
]. The ability of flavonoids to scavenge free radicals and block lipid-peroxidation raises the possibility that TCE extract may act as protective factors against radiation mediated DNA damage. Correspondingly, a number of plant extracts have also been reported to react with free radicals and modulate free radical-mediated reactions mainly through their polyphenolic and flavonoid composition as reported by Prabhakar et al. [71
] and Lia et al. [72
Moreover, supplementation of TCE might have accountable for the increased concentration of phytoantioxidants which seem to be a responsible aspect for lowering the lipid-peroxidation because the basic cause of lipid-peroxidation is not only the free radicals but also the low levels of antioxidants that scavenge them. Alternatively, TCE might have increased the intracellular level of reduced glutathione, and stimulated the immune systems which could have provided protection against the radiation-induced mortality. Since significant radioprotection is obtained at a nontoxic dose of T. cordifolia, it may have an advantage over the contemporary radioprotector available at experimental level. Further, the studies are required to unravel the underlying mechanism of such plant against ROS-mediated damage for improving its efficiency better.