Volume 112, Issue 3 p. 186-191
Original Article
Free Access

Protection by Hydrogen Against Gamma Ray-Induced Testicular Damage in Rats

Zhitao Jiang

Zhitao Jiang

Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China

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Bo Xu

Bo Xu

Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China

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Mowen Yang

Mowen Yang

Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China

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Zhaozhu Li

Zhaozhu Li

Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China

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Yubo Zhang

Yubo Zhang

Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China

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Dapeng Jiang

Corresponding Author

Dapeng Jiang

Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China

Author for correspondence: Dapeng Jiang, Department of Pediatric Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China (fax + 86 451 86605247, e-mail [email protected]).Search for more papers by this author
First published: 23 September 2012
Citations: 19

Abstract

The aim of this study was to investigate the possible protective role of hydrogen-rich saline solution (HRSS) and WR-2721 on the testicular damage induced by irradiation. Sprague-Dawley rats were randomly divided into four groups. Group I served as control group. Rats in group II were exposed to the irradiation. The animals in group III and IV were injected intraperitoneally with HRSS (5 ml/kg) and WR-2721 (200 mg/kg), respectively, 15 min. before the start of gamma irradiation. Testis weight, testis dimensions, sperm count, sperm motility, apoptosis index and biochemical assays were assessed after a 4-day initiation of irradiation. Testis weight, testis dimensions, sperm count, sperm motility in group II were significantly lower compared with those in the control group, whereas they were higher in the HRSS and WR-2721 group. Apoptosis index was significantly increased in group II. Treatment of rats with HRSS and WR-2721 significantly reduced the apoptosis index. On the other hand, irradiation markedly decreased activities of SOD. Activities of SOD were significantly improved when treated with HRSS and WR-2721. Significant increase in the MDA level was observed in group II. MDA levels of group III and IV were significantly lowered when compared with group II. HRSS also played a significant role in the recovery of serum testosterone levels. The results from this experimental study suggest that hydrogen has a possible protective effect against radiation-induced testicular damage.

Radiotherapy has been used increasingly in the treatment of haematological malignancies and solid tumours. Although the treatments could be successful, patients often complain of azoospermia or infertility 1. The testis, an important organ of the male reproductive system, is appreciably radiosensitive because of the presence of rapidly proliferating cells, spermatogonia. It can lead to biochemical and morphological changes caused by irradiation in testicular tissue 2-4. Research on the biological effects of irradiation and the prevention of its inherent deleterious effects in people who have been irradiated is lacking. Knowledge regarding protection of patients from the side effects of radiotherapy, therefore, has become more important.

Irradiation has been shown to decrease ability of spermatogenesis, alter the production of various hormones and induce infertility 2, 5. Spermatogonia are especially sensitive to irradiation. The deleterious effects of irradiation in biological systems are mainly mediated through the generation of reactive oxygen species (ROS) in cells as a result of water radiolysis 6. ROS cause endothelial damage and germinal cell necrosis through interaction with proteins, lipids and nucleic acids causing cellular dysfunction and even death cell membrane lipid peroxidation (LPO), protein denaturation and DNA impairment 7.

Hydrogen (H2) has been considered as a potent free radical scavenger and has been demonstrated to have effective protection against tissue damage such as myocardial injury induced by irradiation 8. Its protective effect is largely due to its ability to limit lipid oxidation and apoptosis 9-11. However, no study has investigated the role of H2 in testicular injury induced by irradiation. Based on these facts, we hypothesized that hydrogen-rich saline solution (HRSS) is one of the reasonable ways to protect testicular damage induced by irradiation. In this study, we investigated the potential radioprotective effects of HRSS and compared its effect with that of WR-2721 (amifostine) in the treatment of testicular damage induced by irradiation in rats using biochemical and histopathological parameters.

Materials and Methods

Animals

All rat experiments were approved by the Animal Ethical Committee at Harbin Medical University. A total of 40 male Sprague-Dawley rats weighing between 150 and 180 g were used in this study. The animals were housed in individual cages with free access to food and water under a 12-hr light: dark cycle. A 1 month period of acclimatization was allowed before any experimental procedures.

HRSS preparation

Hydrogen was dissolved in normal saline solution for 6 hr under high pressure (0.4 MPa) to a supersaturated level. The saturated hydrogen saline was stored under atmospheric pressure at 4°C in aluminium pouches with no dead volume.

Irradiation

60Co-gamma irradiation therapy machine (GWGP80, China Nuclear Power Research Institute, Chengdu, China) was used for the irradiation purpose. Animals were placed in perforated plastic bottles for irradiation individually, and their movements were restricted by anaesthetization. The animals were exposed to 10 Gy gamma irradiation at a dose rate of 1.59 Gy/min. at a distance of 65 cm from the source. Radiation was given to an area of 5 × 5 cm of the scrotum in a supine position. Ten Gray dose was used in the study according to previous work 12.

Experimental design

The animals were randomly divided into four groups, with 10 rats in each group. The groups consisted of a control group, a gamma-irradiated group, a gamma-irradiated + HRSS group and a gamma-irradiated + WR-2721 group. Rats in the control group received neither radioprotector nor irradiation, but saline (5 ml/kg) was injected intraperitoneally. This dosage was based on previous studies 10, 13. The animals in the gamma-irradiated group were injected intraperitoneally with saline (5 ml/kg) 15 min. before the start of irradiation exposure. In the gamma-irradiated + HRSS group and gamma-irradiated + WR-2721 group, HRSS (5 ml/kg) and WR-2721 (200 mg/kg) were injected intraperitoneally into rats 15 min. before the start of exposure to gamma irradiation, respectively.

Sample collection

On the day of surgery, the animals were killed by cervical dislocation after a 4-day initiation of irradiation. The testes were removed, cleared of adhering connective tissue and measured for weight, length and width. The specimens of all groups were used for histopathological examinations and biochemical assays.

Evaluation of sperm count and motility

The epididymal tissues were weighed, minced finely with anatomical scissors in 6-ml phosphate buffer (pH 7.4) and homogenized for 30 sec. Tissue homogenate supernatant was incubated at 37°C for 10 min. to promote the release of sperm in the solution. The suspension was dispersed and filtered to exclude large tissue fragments. Total sperm counts were determined with a haemocytometer under a light microscope at 200× magnification. The sperm count was expressed as sperm per millilitre. Spermatozoa were collected with a micropipette to a slide for motility estimation and were classified into motile and non-motile sperms. At least 400 spermatozoa per group were observed for motility analysis. Motility was evaluated visually at 400× magnification, and data were expressed as percentages.

Detection of apoptosis

The testicular specimens were individually fixed in Bouin's fluid, sectioned and stained. Apoptotic nuclei in tissue sections were identified using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling) assay. The examination of the slides was carried out by a pathologist in a blinded fashion. Only circular tubular cross-sections cut in boldface were studied. Quantitative evaluation of apoptotic index was performed by manual count of the mean number of TUNEL-positive cells per seminiferous tubules per group. The apoptotic cells in the tubules were recorded using light microscopy at the magnification of 400×.

Biochemical assays

Testis tissues were homogenized with buffer containing 1.15% KCl. Malondialdehyde (MDA) concentrations in tissue homogenate supernatant were measured by colorimetric assay based on the interaction of the barbituric acid with MDA. The absorbance was measured at 532 nm. Results were expressed as nanomoles of MDA per milligram protein. Superoxide dismutase (SOD) activity was measured by reduction in nitroblue tetrazolium by xanthine-xanthine oxidase system. One unit of SOD was defined as enzyme activity leading to 50% inhibition.

Measurement of blood levels of LH, FSH and testosterone

Trunk blood was collected and stored for 16 hr at 4°C before separation of the serum by centrifugation for 30 min. at 1500 × g. Serum was stored at −20°C prior to the measurement of LH, FSH and testosterone. The levels of serum LH, FSH and testosterone were determined using traditional radioimmunoassay methods and were performed according to the manufacturer's instructions. All samples from the same experiment were measured in the same assay.

Statistical analysis

Data are presented as mean ± standard deviation (S.D.). One-way anova with the Student–Newman–Keuls test was used for multiple comparisons. Statistical analyses were carried out with spss 11.0 statistical package (SPSS Inc., Chicago, IL, USA). All values of < 0.05 were accepted as statistically significant.

Results

Testis length, width and weight

The values of testis weights and dimensions are shown in fig. 1. The testis dimensions in the gamma-irradiated group, the gamma-irradiated + HRSS and the gamma-irradiated + WR-2721 group showed significant differences in comparison with the control group. Irradiation significantly decreased the testis lengths and widths in comparison with control (p < 0.01) (fig. 1A,B), whereas, when rats were treated with HRSS or WR-2721, there were significant increases in testis lengths and widths in comparison with the gamma-irradiated group (p < 0.01) (fig. 1A,B). Testicular irradiation resulted in significant decreases in testis weights in relation to the individual body weights. HRSS and WR-2721 treatment significantly (< 0.01) restrained the decrease in testis weight per body weight in comparison with irradiated animals (fig. 1C).

Details are in the caption following the image
Effects of gamma irradiation, HRSS and WR-2721 treatment on (A) testis length, (B) width and (C) weight (g)/body weight measured 4 days after initiation of irradiation. Data are shown as mean ± S.D. of 15 separate determinations in each group. *Significant difference from the corresponding value seen in rats from the control group (< 0.01). #Significant difference from the corresponding value seen in rats treated with gamma irradiation (< 0.01).

Sperm count and motility

The number of sperm cells in the caudal epididymis was found to be 19.1 ± 2.1 million/ml in control animals. It significantly decreased to 14.2 ± 1.7 million/ml after exposure to irradiation. The sperm count was elevated significantly (< 0.01) by treatment with HRSS and WR-2721 when compared with the gamma-irradiated group (fig. 2A). Effects of HRSS and WR-2721 treatment on sperm motility had similar effect pattern (fig. 2B). Irradiation had significantly lower (< 0.01) sperm motility compared with the control group. Sperm motility in the gamma-irradiated + HRSS and gamma-irradiated + WR-2721 group was increased by 18.9% and 19.3, respectively, as compared with the gamma-irradiated group (< 0.01).

Details are in the caption following the image
Effects of gamma irradiation, HRSS and WR-2721 treatment on (A) sperm count, (B) sperm motility and (C) apoptotic germ cell indices measured 4 days after initiation of irradiation. Data are expressed as mean ± S.D. of 15 separate determinations in each group. *Significant difference from the corresponding value seen in rats from the control group (< 0.01). #Significant difference from the corresponding value seen in rats treated with gamma irradiation (< 0.01).

Evaluation of germ cell apoptosis

The findings of TUNEL-positive cells in all groups are represented in fig. 3. Quantitative assessment of germ cell apoptosis index is shown in fig. 2C. A small number of seminiferous germ cells exhibited apoptosis in the control group. Testicular irradiation significantly increased the expression of the apoptotic nuclei in the germ cells compared with control testis (< 0.01) (fig. 2C). It is interesting that treatment of rats with HRSS and WR-2721 resulted in significant decrease in programmed germ cell death in the irradiated testis (< 0.01) (fig. 2C), compared with the gamma-irradiated group.

Details are in the caption following the image
Effects of gamma irradiation, HRSS and WR-2721 treatment on apoptosis of germ cell. Testicular tissues in the control group showed fewer apoptotic nuclei. Gamma-irradiated group had a significantly increased number of apoptotic germ cells. Treatment of rats with HRSS and WR-2721 significantly reduced the number of apoptotic nuclei (original magnification × 400).

Biochemical results

The results of testicular MDA levels and SOD activities in all groups are shown in fig. 4. The MDA level in the gamma-irradiated group was significantly increased as compared with the control group (< 0.01) (fig. 4B). Treatment of rats with HRSS and WR-2721 significantly decreased the tissue MDA levels. In gamma-irradiated rats, the SOD activities were significantly decreased when compared with the control group. SOD activities of rats in the gamma-irradiated + HRSS group and the gamma-irradiated + WR-2721 group were higher than those of the irradiated rats and were close to the level of the control rats, indicating that HRSS or WR-2721 treatment may be useful for scavenging oxygen-derived free radicals.

Details are in the caption following the image
Effects of gamma irradiation, HRSS and WR-2721 treatment on (A) activities of SOD and (B) MDA levels measured 4 days after initiation of irradiation. Data are shown as mean ± S.D. of 15 separate determinations in each group. *Significant difference from the corresponding value seen in rats in the control group (< 0.01). #Significant difference from the corresponding value seen in rats treated with gamma irradiation (< 0.01).

Serum hormone levels

Comparing the irradiated rats with the control rats, the average levels of testosterone were significantly lower. Administration of HRSS or WR-2721 before gamma irradiation significantly rescued testosterone levels. Levels of FSH as well as of LH were not significantly different in all groups (fig. 5). Failure to detect the alteration in FSH and LH may be attributable to the fact that in the present experiments the rats were killed at only 4 days.

Details are in the caption following the image
Effects of gamma irradiation, HRSS and WR-2721 treatment on (A) LH, (B) FSH and (C) testosterone levels measured 4 days after initiation of irradiation. Data are shown as mean ± S.D. of 15 separate determinations in each group. *Significant difference from the corresponding value seen in rats in the control group (< 0.01). #Significant difference from the corresponding value seen in rats treated with gamma irradiation (< 0.01).

Discussion

Irradiation has been used increasingly in medicine and industry to help with diagnosis, treatment and technology. Irradiation causes a disturbance in the normal metabolism, proliferation and differentiation, which may lead to mutagenesis, apoptosis and necrosis of radiosensitive cells. Testicular spermatogenic cells are highly sensitive to irradiation, and temporary or even permanent infertility is a common post-irradiation problem. Discovering effective agents with radioprotective effects is important for protecting patients from the side effects of radiotherapy.

Irradiation could induce the body to generate large amounts of oxygen-free radicals 14. It is generally believed that oxidative stress affects the organism when the generation of ROS products exceeds the capacity of the cells to protect or repair themselves 15. ROS can cause LPO in the cellular and mitochondrial membranes. Peroxidation of the lipids in membranes changes membrane permeability or disrupts membrane integrity and causes DNA damage of germ cells 15. DNA damage caused by irradiation in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa 16. Irradiation exposure can also alter the balance of endogenous protective systems such as glutathione and antioxidant enzyme systems 17.

A recent study has revealed that H2 has protective properties against irradiation – induced lung damage by reducing oxidative stress 18. It can also attenuate brain, intestinal and myocardial ischaemia/reperfusion injury, and this protective effect is largely due to its ability to limit lipid oxidation 9, 13, 19. Although H2 has been investigated for multiple purposes, its effect on injury of the testes caused by irradiation has not yet been evaluated. In this study, we aimed to evaluate the effects of H2 on the irradiation-induced testicular damage in comparison with that of WR-2721. We measured a variety of parameters related to testicular damage after administration of hydrogen-rich saline.

The weight of testes is largely dependent on the mass of differentiated spermatogenic cells. Irradiation affected cell proliferation, depletion of spermatogonial cells and their subsequent generations, leading to loss of testis weight 20. In this study, the testis width and testis/body weight ratio of rats were significantly reduced after exposure to irradiation, in agreement with an earlier report. The testis width and testis weight per body weight were significantly improved when HRSS and WR-2721 were administered. We observed an apparent protecting effect of HRSS against irradiation-induced injury of testis.

Sperm count and motility are important indicators for reflecting spermatogenic capacity and predicting male reproductive function. Investigating sperm count and motility is important to evaluate the damage caused by irradiation 21. In our study, it was found that when testicular tissues were exposed to irradiation, sperm quantity and motility were significantly decreased. HRSS significantly improved recovery of sperm quantity and motility, which is a similar effect to that observed for WR-2721.

The loss of male germ cells after exposure to irradiation has been attributed to apoptosis 22. In this study, we also found that irradiation of testes induced apoptosis of germ cells. Apoptosis index was increased in the gamma-irradiated group when compared with that of the control group. A previous study had demonstrated that HRSS exerts an anti-apoptotic effect on cardiac cells 19. In our study, it was found that HRSS remarkably inhibited germ cell apoptosis after irradiation. Also, the effect of HRSS was similar to the effect observed for WR-2721. All of those findings were statistically significant. We histologically confirmed the protective effect of HRSS on injury of the testes caused by irradiation.

Malondialdehyde is a by-product of lipid peroxidation and is widely used as a reliable marker of tissue damage. In our study, we observed that intraperitoneal injection of HRSS before irradiation significantly decreased MDA concentration in rats exposed to the irradiation. SOD is a key component in removing superoxide radicals and preventing the damage caused by free radicals. The antioxidant effect of H2 is supported by the finding in this study that hydrogen can protect testis from radiation-induced injury via increasing endogenous SOD. Our results demonstrate that the administration of HRSS protects the testes against free radical generation, which is similar to that of WR-2721.

Various forms of radiation could cause sex hormone disorder. Testosterone is a key hormone secreted by the testis that promotes spermatogenesis 23. The results showed that serum testosterone levels decreased significantly after local irradiation. Treatment with HRSS and WR-2721 restored testosterone levels. It indicated that HRSS maintains the spermation functions of testicles by keeping balance of serum sex hormone. Irradiation had no effect on the serum FSH, and LH levels may be attributable to the fact that the rats were killed at only 4 days in this study.

Taking these data together, we conclude that hydrogen acted as a good scavenger against free radical damage and thereby maintains functional integrity of the cell, spermatogenesis and sperm motility. This study examined the effect of HRSS on testicular injury after irradiation at only one time-point; therefore, kinetic effects are unknown and need to be investigated in future studies. Although more work needs to be performed, present results provide evidence that HRSS has useful clinical applications in the prevention of testicular injury after gamma irradiation. For this purpose, further clinical studies are needed.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No.30901516), Reserve Talents of Universities Overseas Research Program of Heilongjiang and Natural Science Foundation of Heilongjiang (QC2011C049).

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