INTRODUCTION

Carbon tetrachloride is used extensively in experimental models to induce oxidative stress in rats (Onori et al., 2000; Nabeshima et al., 2006; Noori et al., 2009). A single dose of CCl₄ can rapidly lead to both oxidative stress and acute liver injuries such as centrilobular necrosis and steatosis in rats (Weber et al., 2003; Lin et al., 2008; Khan and Alzohairy, 2011). Previous data demonstrated that rats with advanced liver cirrhosis showed reduced testicular size and weight and severe histopathological testicular abnormalities, including reduced tubular diameters, loss of the germinal line and diminutions in cellular proliferation and spermatogenesis (Castilla-Cortazar et al., 2000). Also previous data demonstrated that rats treated by CCl₄ showed histopathological testicular abnormalities and loss of the germinal line (Khan and Ahmed, 2009).

The biological effects of low-level radiation have attracted the attention of investigators for more than 20 years. The biological effects of low-level radiation on cellular metabolisms and defence systems sometimes called hormesis (Feinendegen, 2005) by increased immune responses and antioxidant capacity (Lee et al., 2000; Gong et al., 2000; Joksic and Petrovic, 2004). However, Liu et al. (2006) reported that the exposure of the experimental animals to low-level radiation induces increased apoptosis in male germ cells. Bone marrow transplantation (BMT) is increasingly used in the therapy of solid malignancies, as well as in non-malignant disorders such as thalassemia and immunodeficiency (O’Reilly, 1983; Champlin and Gale, 1987). Also the preclinical and clinical studies have demonstrated that bone marrow stromal cells (MSCs) can be used for tissue repair (Yoon et al., 2005). The successful application of Bone Marrow Transplantation BMT to the treatment of several potentially fatal disorders (Storb et al., 1976; Powles et al., 1980; O’leary et al., 1983) has resulted in a variety of late complications on gonadal function and future fertility.

The present study was done to recognize the effect of exposure to low dose of γ-radiation on testis tissue in rat treated CCl₄ model and the role of BM transplantation.

MATERIALS AND METHODS

Experimental animals: Male Swiss albino rats (100-120 g) purchased from the Egyptian Organization for Biological Products and Vaccines were used for the different investigations carried out in this work. Animal maintenance and treatments were conducted in accordance with the National Institute of Health Guide for Animal, as approved by Institutional Animal Care and Use Committee (IACUC). Animals were housed in specially designed cages and maintained in conditions of good ventilation, normal temperatures and humidity ranges and kept under observation for one week prior to experimentation. The rats were fed on standard pellets, containing all nutritive elements (proteins, fats, carbohydrates, vitamins, salts and minerals). Drinking water and food were provided ad libitum throughout the study.

Radiation facility: Whole-body γ-irradiation was performed at the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt, using an AECL Gamma Cell-40 biological irradiator. Animals were irradiated at dose level of 0.5 Gy 2 times/week for 8 weeks. The γ-irradiation delivered at a dose rate of 0.46 Gy/min.

Rat bone marrow preparation: Donors and recipients rats were chosen of the same inbred strain, brother to brother (isologues or synergic or allogeneic transplantation). Rats sacrificed by exposure to ether in a dessicator kept in a well-functioning hood. Femur bones were dissected out and cleaned. The ends of the bones were chipped by a bone nibbling forceps. Then the marrow was blown out of the femur into isotonic solution under sterilized conditions inside a laminar flow cabinet. The marrow was collected into a sterile container surrounded by ice cubes and mixed by drawing and expelling it several times from the syringe without needle in order to avoid mechanical damage to the cells. Total viable cells of about 75x106±5% were injected intravenously (IV) through the caudal vein. All Rats treated with bone marrow cells transplantation were killed after four weeks of bone marrow cells transplantation.

Carbon tetrachloride administration: Rats were intraperitoneally injected with 1 mL kg^-1 of carbon tetrachloride (CCl₄) dissolved in olive oil (1:1) twice a week for 8 weeks.

Experimental design: A total of 48 rats were divided into the following sub groups.

Non Bone marrow administrated groups including:

Bone marrow administrated groups including:

Histopathological examination: Excised liver and testis tissues from each rat were fixed in 100 mL L^-1 neutral formalin, embedded in paraffin and stained with hematoxylin-eosin (HE) and the fibrous lesion areas were determined via Masson's trichrome method which is used to stain collagen fibers.

Apoptosis and necrosis examination: For apoptosis and necrosis examination according to Bank (1988) fluorescence microscopy was used. Deparaffinization was done by immersing tissue sample slides in 3 changes of xylene for 5 min each followed by washing in graded alcohol as follows: 100, 95, 80 and 50% for re-hydration. Two changes for 3 min in each alcohol concentration were done. Then, they were rinsed in 3 changes of PBS. Afterwards, slides were directly incubated in (5 μg mL^-1 of propidium iodide and 50 μg mL^-1 of acridine orange in phosphate-buffered saline) in dark for 20 min at room temperature.

Analysis of biochemical assay: Serum obtained from the blood samples were analyzed for aspartate aminotransferase (AST) (Bergmeyer et al., 1985), alanine aminotrasnferase (ALT) (Klauke et al., 1993) and total protein (Keller, 1984) by using kit purchased from Stanbio (USA). However, serum testosterone and estradiol ware quantitatively determined in the sera by enzyme immunoassay kit (Meddix Bioech Inc, 420 Lincoln Centre Drive, Foster City, CA 94404, USA, Catalog Number: KEF4057).

Statistical analysis: Statistical analysis for obtained results was carried out with the aid of the SPSS computer software program.

OBSERVATIONS AND RESULTS

At baseline, in Table 1 compared to control level (p<0.05) CCl₄ treated group showed a significant increase in serum ALT and AST levels but total proteins represented, no significant change. Exposure of control rats to fractionated low dose of γ-radiation recorded a no significant change in serum ALT and AST levels but total proteins represented, a significant increase compared to control level (p<0.05). On the other hand exposure of CCl₄ treated group to fractionated low dose of γ-radiation recorded a decrease in serum levels of ALT, AST but total proteins level recorded a significant increase in compression to CCl₄ group level (p<0.05). Bone marrow transplantation in control animals represented a significant decrease in serum ALT, a significant increase in serum AST and no significant change in total proteins. However, BM transplantation in CCL₄ or CCl₄ R groups showed ameliorating effect in AST, ALT and total proteins levels compared to CCl₄ treated group.

Histopathologically liver tissue sections in rats suffered from CCl₄ treatment (two times per week) for eight weeks showed hepatocytes degeneration, necrosis, mononuclear cells and neutrophil infiltration. Also, collagen fibers extend within the hepatic plate was observed. Bone marrow cells transplantation showed normal hepatic tissue section. Exposure of the CCl₄ treated group to 0.5 Gy (two times per week) for eight weeks showed some sort of regeneration in a considerable number of hepatocytes, inhibition of inflammatory cellular infiltration in many areas and less prominent of cytoplasmic vacuolation.

Table 1:	Effect of CCl4, radiation and BM either alone or combined on liver functions
Values are given as mean ±SD (n = 6), *Significantly different from control at 0.05, #Significantly different from CCl4 treatment at 0.05

On the other hand liver section in CCl₄ (two times per week for eight weeks) rats irradiated with 0.5 Gy and treated with bone marrow cells (one time at the fourth week) transplantation recorded that bone marrow cells helped very much in regain of most hepatocytes cellular structure. There are no signs of cell weakness as many hepatocytes appeared with well defined membranes, homogenous cytoplasm and healthy normal nuclei. Many mitotic figures were noted, blood sinusoids were normal in size and the blood vessel was well organized.

TESTICULAR TISSUE OBSERVATIONS

H and E stain: In Fig. 1 the light microscopic examination of the testis of control rats showed normal structure and completely enveloped by a thick capsule, tunica albuginea which is composed mainly of dense collagenous fibrous connective tissue. The structural components of the testis are the seminiferous tubules and interstitial tissues. The seminiferous tubules are two types of cells, the Sertoli cells, resting on the thin basal lamina (basement membrane) and the spermatogenic cells. These cells are many layers, namely, the spermatogonia, primary and secondary spermatocytes; spermatoids and finally mature spermatozoa. Treatment of the experimental animals by CCl₄ (two times per week) for eight weeks noted wide interstitial space, seminiferous tubuli in testis appear seriously damaged and animals show a decrease of tubular diameter, vacuolization on germinal epithelium, loss of germinal line, total or partial reduction of spermatogenesis and presence of abnormal spermatids (multinucleated cells and cells with an intense stained nuclei). Exposure of control rats to 0.5 Gy (two times per week) for eight week showed atrophy and decrease in size of seminiferous tubule, tubular profiles completely depleted of germ cells and some hyperplasia in the Leydig cell was observed. Increase in testicular disorganization was detected in seminiferous tubules when CCl₄ (two times per week for eight weeks) treated animals exposed to 0.5 Gy γ-radiations (two times per week for eight weeks).

Fig. 1(a-h):

Microscopy of testes (×400 magnifications, H&E stain). Testicular histological sections of rat demonstrated active spermatogenesis in normal-size seminiferous tubuli with thin basement membranes (▲). Leydig cells were scarce, being widely separated by seminiferous tubuli. BM represented discontinuous seminiferous epithelium (short arrow). Either CCL4 or R exposure alone or combined reflect testicular seminiferous tubules atrophy and completely depleted of germ cells (). BM treatment of CCL4 group reflects great recurrence of seminiferous tubules normal structure in testes. Exudation (E) into the interstitial space and degeneration/necrosis (N) of spermatogenic and great interstitial hyperplasia (long arrow) in R+BM and CCL4+R+BM groups

Table 2:	Effect of CCl4, radiation and BM either alone or combined on testosterone and estradiol testicular hormones
Each value represents the mean of 6 records±SE. Means with different superscripts are significantly different at the 0.05 level.* significatly different from control, #significantly different from CCl4. ! significantly different from CCl4+BM and R+BM

Seminiferous tubules in testis appear seriously damaged, decrease in tubular diameter, vacuolization on germinal epithelium, loss of germinal line, total reduction of spermatogenesis and increase in hyperplasia in the Leydig cell were observed.

Treatment of control group with BM cells represented discontinuous seminiferous epithelium. Great recurrence of seminiferous tubules normal structure in testis when experimental animals treated by CCl₄ (two times per week) for eight weeks and treated by bone marrow cells transplantation (one time at the fourth week) was occurred. Exudation into the interstitial space and degeneration/necrosis of spermatogenic cells were observed when rats irradiated for four weeks and then bone marrow cells transplantation occurred followed by irradiation again for another four weeks. Atrophied testicular seminiferous tubules and great interstitial hyperplasia were shown in testis of CCl₄ irradiated rats for four weeks, followed by bone marrow cells transplantation one time and continue the process of irradiation and CCl₄ treatments for another four weeks.

Masson's trichrome stain: In Fig. 2 treatment of the experimental animals with CCl₄ or low dose of γ- radiation exposure either alone or combined represented peritubular fibrosis or increase in collagen deposition. Treatment of the previous groups with BM represents a remarkable depletion in peritubular collagen deposition with its great deposition on blood vessels sides.

Apoptosis and necrosis observations: In Fig. 3 testicular sections of normal rat (Control) demonstrated anon apoptotic observations in seminiferous epithelium. CCl₄ treatment (two times per week for eight weeks) recorded the presence of apoptotic cells in seminiferous epithelium. Both 0.5 Gy of γ-radiation exposure (two times per week for eight weeks) or CCl₄ and 0.5 Gy irradiation (two times per week for eight weeks) treatments represented apoptosis in the Leydig cells. BM cells transplantation showed many apoptotic observations in the spermatogonia, sertoli cells and primary spermatocytes. Interstitial tissue represented necrotic observations. Treatment of CCl₄ group by BM cells transplantation recorded apoptotic and necrotic cells. Also treatment of irradiated animals either alone or combined with CCl₄ treatments by BM cells transplantation represented many interstitial apoptotic and necrotic tissue cells.

Analysis of testosterone and estradiol testicular hormones: In Table 2 testicular hormonal analysis of testosterone concentration was investigated in this study. A non-significant reductions in testosterone concentration mean values when compared with the control concentration (p<0.05) following CCl₄ treatment (two times per week for eight weeks). However, exposure of control rats to fractionated dose of γ-radiation (0.5 Gy two times per week for eight weeks) recorded a significant reduction in testosterone concentration level when compared with the control level (p<0.05). Combined treatment of the experimental animals by CCl₄ and γ-irradiation recorded a significant decrease in testosterone level compared to control or irradiated group. BM cells transplantation in rat’s empire their testosterone level either alone or following CCl₄ or γ-radiation exposure registered a significant decrease comparing to control level.

Also Table 2 represented Estradiol levels in different studied groups. Estradiol levels showed a significant decrease for the treatment of the experimental rats by CCl₄. Meanwhile radiation exposure represented a significant increase in compression to control level. Combined treatment of CCl₄ and fractionated dose of γ-irradiation (0.5 Gy two times per week for eight weeks) recorded a significant increase in estradiol level compared to control or irradiated group levels. On the other hand BM cells transplantation in experimental rats recorded a non- significant change in estradiol level when compared with the control level (p<0.05) following CCl₄ treatment or radiation exposure.

DISCUSSION

The present study was designed in order to gain more insights into the effect of exposure to consecutive low dose of γ-radiation and bone marrow transplantation were evaluated in CCl₄ treated rats and the altered changes in testis tissue associated with liver disease.

Our study demonstrates that rats treated by CCl₄-induced many histopathological and biochemical altered changes in liver tissue show a severe testicular atrophy and gonadal insufficiency. Both testicular histopathological abnormalities and low levels of sex hormones have been described in previous years in patients with alcoholic and nonalcoholic cirrhosis (Pajarinen and Karhunden, 1994; Van Steenbergen, 1993).

Fig. 2(a-h):	Microscopy of testes (x400 magnifications, Masson’s stain). Testicular histological sections of normal rat (Control) demonstrated minimal peritubular fibrosis. Evidence of peritubular fibrosis and other alterations were found in testes from R, CCl4 and CCl4+R treated animals. Treatment with BM recorded great deposition of collagen fibres (↑) around blood vessels.

Our data show a severe testicular damage as manifested by a variety of histopathological abnormalities that include alterations in tubular diameters, presence of aberrant cells in tubular lumen, peritubular fibrosis, loss of the germinal line and the presence of apoptotic cells in seminiferous epithelium. These alterations resemble those reported in experimental models of testicular damage, such as chronic testicular ischemia (Santamaria et al., 1995; Al-Jahdali and Bisher, 2007).

Fig. 3:

Fluorescent Microscopy of testes (x400 magnifications, propidium iodide/acridine orange stain). Testicular sections of normal rat (Control) demonstrated anon apoptotic observations in seminiferous epithelium. CCL4 treatment recorded the presence of apoptotic cells in seminiferous epithelium (↑). Either Radiation exposure or CCL4 and irradiation treatments represented apoptosis in the Leydig cells (↑). BM transplantation showed many apoptotic observations in the spermatogonia, sortoli cells and primary spermatocytes. Interstitial tissue represented necrotic observations. CCL4BM treatment recorded apoptotic (↑) and necrotic cells (). Treatment of irradiated animals by BM represented many interstitial apoptotic and necrotic tissue cells. Treatment of CCL4 and irradiated group by BM also represented interstitial apoptotic (↑) and necrotic tissue cells ()

The occurrence of testicular atrophy and gonadal dysfunction in advanced cirrhosis is a well-known clinical event (Van Thiel et al., 1980; Bannister et al., 1986; Pajarinen and Karhunden, 1994).

Ranl et al. (2007) proposed that the cause of testicular histopathology may be attributed to the malfunctioning of liver (Lox, 1984) which causes general systemic toxicity due to some toxic factors in peripheral circulation which influence testicular functions.

The non significant change in testosterone level and the significant decrease in estradiol level of CCl₄ treated animals in our study was context with finding of Frezza et al. (1993).

According to Withers et al. (1974) changes in weight and size of the irradiated testis is related more to depletion and regeneration of much more numerous cells in various stages of differentiation and are not a direct indication of stem cell depletion.

Nomura and Yamaoka (1999) proposed that Low-dose gamma-ray irradiation reduces oxidative damage induced by CCl₄ in mouse liver and suggest that low-dose radiation relieved functional disorder at least in the liver of mice with active oxygen diseases.

In the present study γ-irradiation treatments represented great atrophy in testis tissue, pretubular fibrosis and apoptosis in the Leydig cells, context with the finding of Kangasniemi et al. (1996) and Meistrich et al. (1996) who proposed after exposure to low doses of gamma rays differentiating spermatogonia are killed. The depletion in spermatogonia resulted in a reduction in subsequent spermatozoa. Also, Konoplia et al. (1996) examined the microscopy morphological characteristics of Sertoli cells. Leydig's cells and other populations of testicular cells after prolonged of low dose whole-body gamma-irradiation and suggests that the existence of gamma-sensor in brain of mammals that involved on hypothalamic-pituitary-testicular levels in realisation of radiation stress suppression of Sertoli cell functions at a relatively "low" (0.1-0.5 Gy) doses by means of hypothalamic releasing factors.

Isao et al. (2004) proposed that mice with BMC transplants with continuous CCl₄ injection had reduced liver fibrosis and a significantly improved survival rate after BMC transplantation compared with mice treated with CCl₄ alone. This finding introduces a new concept for the therapy of liver fibrosis.

In the present study BM transplantation empire testicular hormones, many apoptotic and necrotic observations in the spermatogonia, sertoli cells and primary spermatocytes and discontinuous seminiferous epithelium context with the finding of Yong-Hoon et al. (2009). Also, the same observations were recorded when CCl₄ or CCl₄ irradiated group treated by bone marrow transplantation. Autoimmune-like complication after BMT leads morphological and functional changes of target tissues (Levy et al., 2000) and also associated with gonadotoxicity (Wagner et al., 2005).

In conclusion exposure to consecutive low dose of γ-radiation and bone marrow transplantation impair testicular tissue in a rat treated CCl₄ model.