Abstract
Different forms of Aluminium (Al) are environmental xenobiotics that induce free radical–mediated cytotoxicity and reproductive toxicity. Vitamin E (α-tocopherol) is an antioxidative agent that has been reported to be important for detoxification pathways. This study was thus aimed at elucidating the protective effects of vitamin E towards aluminium toxicity on the histology of the rat testis. Al (5 mg/kg body weight) was administered intraperitoneally in 2 ml saline, either alone or immediately before vitamin E (500 mg/kg body weight), at a different point of abdomen, and the alterations in the testis tissue were analyaed histologically. Seven treated animals were sacrificed for each group, with the testes removed and examined histologically. In the Al-treated group, the germinal epithelium of the seminiferous tubules was thinner in places and spermatids were almost absent; sperm numbers were low and there were no sperm in the lumen. In the Al plus vitamin E rats, there were large numbers of spermatids and sperm in the seminiferous tubule lumen. In the vitamin E alone group, a normal histology was seen. Electron microscopically, in the Al-treated group there were irregularities in the nuclear membrane, some damaged mitochondria, a decrease in the number of ribosomes, and an increase in the number of lysosomes in the sertoli cell cytoplasm. In the primary spermatocyte cytoplasm, there was an increase in the rough endoplasmic reticulum. In the Al plus vitamin E group, the spermatogeneic cells and the sertoli cell cytoplasm showed an almost normal appearance. The ultrastructure of the testis in the vitamin E alone group showed a normal appearance. In conclusion, vitamin E antagonizes the toxic effects of Al at the histological level, thus potentially contributing to an amelioration of the testis histology in the Al-treated rats. The associated biochemical parameters merit further investigation.
Keywords
The different forms of aluminum (Al) have been shown to be systemic toxicants (Exley et al. 1996), and there is now considerable evidence demonstrating that the accumulation of Al in body tissues is associated with damage to the target organs (Becaria, Campbell, and Bondy 2002). Al accumulation is most pronounced in humans through high-Al dialysate (D’Haese et al. 1999), but it can also occur due to dietary, occupational, cosmetic, medication, and adjuvant exposure (Popinska et al. 1999; Flarend et al. 2001).
For the male reproductive system, Al is a potential toxicant, as it reduces the weight of the testes and causes decreased epididymal sperm counts in the mouse (Llobert, Colomina, and Sirvent 1979). High concentrations of Al in human spermatozoa and seminal plasma correlate with decreased sperm motility and viability (Schrag and Dixon 1985; Hovatta et al. 1998; Guo, Lu, and Hsu 2005b). Spermatidcell and spermatozoa necrosis and significant decreases in fertility have also been seen in mice exposed to Al (Guo et al. 2001); these previous studies also demonstrated the toxicity of accumulated Al on testicular testosterone production in male mice (Guo et al. 2005a). Many of the heavy metals have also been shown to be harmful with regard to testicular function and sperm production, thus acting as toxicants (Llobet, Colomina, and Sirvent 1979; Guo et al. 2006).
In biological systems, increased oxidative stress has been related to increasing levels of nitric oxide (NO) products. NO is generated from l-arginine by the enzyme nitric oxide synthase (NOS), and it is formed in a variety of tissues and involved in many physiological and pathological processes (Moncada, Palmer, and Higgs 1991). The finding that NOS is also present in animal testes and in other reproductive organs of the human reproductive system (Burnett et al. 1995; Davidoff et al. 1997) suggests that NO may mediate the biosynthesis and secretion of the steroid hormones, and thus may affect the functions of the interstitial cells in the testes (Welch et al. 1995). High levels of NO products have also been seen in many high-dose Al–induced testicular injuries (Guo et al. 2002), and NO-related agents can suppress testosterone secretion in male rats (Adams et al. 1994).
In addition to these, and in spite of the low gastrointestinal absorption, the accumulation of very low nontoxic doses of AlCl3 alone over a long period of time can result in dose-dependent neurotoxic and nephrotoxic effects (5 to 20 mg/kg body weight/day for 6 months) (Somova, Missankov, and Khan 1997). Various forms of Al have different gastrointestinal absorption rates. Al sulphate (Al2(SO4)3) is generally used in animal experiments owing to its high absorption rate. Similarly, different forms of Al can have different accumulation and excretion rates and toxicities. Therefore, rats respond differently to Al chloride and Al citrate, as the kidneys of rats following intravenous administration of Al citrate were seen to contained much higher concentrations of Al than those rats that received Al chloride (Spencer et al. 1995). Significant accumulation of Al has also been shown in liver and kidney, where a long-term intravenous model of Al maltol administration showed lower Al levels in heart and other tissues (Bertholf et al. 1989). When Al was given as the citrate, the urinary excretion was significantly higher than when it was given as the chloride or sulphate (Lote et al. 1995). However, despite many studies on Al toxicity, the mechanisms of Al-induced damage remain unclear.
Ascorbic acid is an antioxidative agent that has been shown to protect the rat testis from deleterious oxidative effects of Al. The antioxidative agent used in the present study, vitamin E (α-tocopherol), is involved in the regulation of testosterone biosynthesis in spermatogenesis, and has been seen to have protective effects against impairment resulting from oxidative agents (Yousef, El-Morsy, and Hassan 2005).
There have been a few studies in the literature describing the absorption of vitamin E following its oral intake. Vitamin E excretion in the feces is increased significantly with increased vitamin E intake, and the absorption rate of vitamin E appears to be inversely proportional to the level of long-term oral exposure (Elmadfa and Walter 1981). In addition, free tocopherol (as α-or γ-tocopherol) absorption requires the presence of bile salts (Traber et al. 1986).
There are three specific cases where a vitamin E deficiency may occur: in people who are not able to absorb dietary fat; in premature, very-low-birth-weight infants (birth weights less than 1.5 kg, or 3.5 lb); and in individuals with disorders of fat metabolism. Therapeutic doses of vitamin E by both parenteral and oral routes are the same in humans, with the average human daily intake being 0.1 to 17.5 μg (Kohlschütter et al. 1988).
The goal of the present study was thus to investigate the Al-induced damage and the potential protective effects on these of the antioxidative agent vitamin E on male rat testis histology. Specific doses of Al were administered through an intraperitoneal route, with the inclusion of the further control of vitamin E alone.
METHODS
Adult male albino Wistar rats were housed in the Experimental Research Unit, University of Pamukkale, Denizli, Turkey. They were reared under the supervision of a veterinarian, kept in a well-ventilated, noiseless environment, and allowed free access to food and water. The temperature was maintained at 21°C, with 50% humidity and a 12-h light-dark cycle. The cages were 40 cm in length, 30 cm in width, and 15 cm in height The male animals (180 to 200 g) were divided randomly into three groups, each of which included seven rats.
In the first, control, group, the animals were injected intraperitoneally with saline (10 ml/kg body weight) using an insulin syringe. The second group were Al treated, by intraperitoneal injection of Al2(SO4)3 (5 mg/kg body weight) diluted in saline (10 ml/kg body weight). To determine the potential protective effects of vitamin E on Al damage to the testes, the third group were injected with both the Al2(SO4)3 (5 mg/kg body weight) and vitamin E (500 mg/kg body weight) diluted in saline (10 ml/kg body weight). These were injected one immediately after the other, and at different points in the abdomen. The injections were performed intraperitoneally over a period of 2 weeks, at 3 times per week (Mondays, Wednesdays, and Fridays); at the end of the second week, the animals were sacrificed by decapitation under anesthesia. The testis tissue was collected from each animal.
Light Microscopy
Some of the testicular tissue from each of the seven rats from the three groups was processed with metachrylate (Technovit 7100, UK), embedded, and sectioned for routine histology following hematoxylin and eosin staining. This included xylene treatment (30 min) and 5-min incubations in decreasing levels of ethyl alcohol (96%, 85%, and 50%), followed by hematoxylin (5 min), acidic alcohol, ammonia, eosin (5 s), and increasing levels of alcohol (50%, 85%, and 96%). Following a further xylene incubation, the samples were covered with coverslips with DPX mounting medium (Technovit, UK). They were later analyzed under light microscopy and photographed.
Electron Microscopy
The remaining testis tissue from each of the rats of the three groups was dissected out and cut into approx 1-mm3 cubes. These were fixed in gluteraldehyde-containing phosphate buffer, pH 7.2, and then post-fixed in a 1% OsO4 solution, for 1 h at 4°C. The samples were dehydrated through increasing ethanol concentrations (75%, 85%, 96%) following this fixation, and then embedded in araldite. Finally, thin sections were stained with uranyl acetate and lead citrate for examination and photography (Jeol 906 E transmission electron microscope).
The study was approved by the Pamukkale University Ethical Committee for Experimental Animals.
RESULTS
Light Microscopy
The seminiferous tubules in the control group had a normal appearance, containing abundant amounts of spermatids and sperm in the lumen (Fig. 1A ). Under greater magnification, there were spermatogonia, primary and secondary spermatocytes and spermatids based on the basal membrane. Mitotic activity was also seen in the germinal cells (Fig. 1B ).
In the Al-treated group, the germinal epithelium was thinner in the places where the spermatids were absent, and the sperm numbers in the lumen were particularly low. Here, there were some unidentifiable cells, which were free in the lumen of the seminiferous tubules (Fig. 1C ). Under higher magnification, although there was mitotic activity and spermatids, the germinal epithelium was particularly thin in places, and spermatogonia were only seen on the basal membrane; there were no sperm in the lumen and spermatid numbers were low. Some of the germinal cells appeared to have lost their cytoplasm, and the cells that had picnotic nuclei had become necrotic. The tight junctions were also broken down and there were cells free in the lumen (Fig. 1D ).
In the Al plus vitamin E rats, although there were similar thin basal membrane areas to the Al-treated animals, there were large numbers of spermatids and sperm. Free cells in the lumen were very rare (Fig. 1E ). Under higher magnification, there was apparent mitotic activity in the germinal cells and there were spermatids in the areas near the lumen (Fig. 1F ).
Electron Microscopy
Under electron microscopy, the seminiferous tubule sections of the control group showed a normal histology (Fig. 2A ). In the Al-treated group, there were some enlarged mitochondria in the sertoli cell cytoplasm, some of which displayed a hole with an electron-dense ring. In the sertoli cell cytoplasm, there were also myelin-like structures. In the primary spermatocyte cytoplasm, there was an increase in the rough endoplasmic reticulum (Fig. 2B ). In some samples from these Al-treated rats, some of the mitochondria showed variations in size and shape, and their cristae showed a tubular or vesicular appearance. Furthermore, in the cisternae of the enlarged rough endoplasmic reticulum, there was a decrease in the number of ribosomes, whereas there was an increase in the number of lysosomes and irregularities seen in the nuclear membrane of sertoli cells (Fig. 2C ). Again, in the samples from the Al plus vitamin E group, the spermatogeneic cells showed an almost normal appearance in these micrographs (Fig. 2D ).
DISCUSSION
Although the rat model may not indeed be directly applicable to humans, the relevance of its use lies in the information that we can obtain at this level of study, and as such, rat models have formed the mainstay of many similar initial toxicity studies (Bondy, Liu, and Guo-Ross 1998; El-Demerdash et al. 2004). In various studies, numerous heavy metals have been shown to have deleterious effects on testicular function and sperm production (Guo et al. 2001; Rao and Sharma 2001; El-Demerdash et al. 2004; Mishra and Acharya 2004). Al has also been seen to be a systemic toxicant, with its ingestion at excessive levels leading to its accumulation in target organs; this has been associated with damage to testicular tissue (Becaria, Campbell, and Bondy 2002; Guo et al. 2005a). Indeed, Guo et al. administered 35/mg/kg/day AlCl3 to mice for 16 days and reported that this Al accumulation led to damage of the testicular tissues. Further studies in mice have also shown signs of increased necrosis in spermatid and spermatozoa, and substantially impaired fertility following Al treatment (Llobet, Colomina, and Sirvent 1979; Hovatta et al. 1998; Guo et al. 2005a, 2005b).
The daily human oral intake of Al has been estimated to be 9 to 14 mg, with pharmacological doses of Al as antacids estimated to be 1 to 3 g daily (Roy, Talukder, and Sharma 1991). The absorption rate of Al via the oral route is low, at 0.1% to 1.0% (Sargazi, Roberts, and Shenkin 2001). Human blood aluminium blood level is 11 μg/100 ml (Martindale 1989). Highest level of 26Al are found in bone (Zafar et al. 1997) in rats. The tissue distribution of Al in rats killed 3 weeks aftert a single intravenous injection of trace amounts of 26Al was found to be 0.9% in bone, 0.2% in kidney, 0.06% in liver, 0.03% in heart, and 0.02% in brain and muscle (Walker et al. 1994). Schteinger et al. (1999) showed that the accumulation of aluminium was in the order liver > kidney > brain > other tissues in mice. In human retained Al originated from aluminium hydroxide is accumulated in bone, liver, muscle, and brain (Clarkson, Luck, and Hynson 1972). The levels of Al in different tissues are Al increases peroxidation and nitric oxide synthase (NOS) activity. This oxidative activity induced by Al nitrate can thus increase NO production in cells (Llobet, Colomina, and Sirvent 1995; Welch et al. 1995; Guo et al. 2005a), and it has been shown that increased oxidative stress and testicular damage can indeed result from Al-induced increases in NO (Guo et al. 2005a). Excessive NO levels have also been shown to suppress testosterone synthesis and to cause cytotoxicity of spermatozoa, with a decreased vitality and mobility of sperm and an increase in morphological abnormalities of sperm. (Llobet, Colomina, and Sirvent 1979; Adams et al. 1994; Bondy, Liu, and Guo-Ross 1998). Thus, as with some of the heavy metals, Al treatment can lead to testicular tissue damage through increased NO production. Similar deleterious effects on testicular tissue by cadmium, iron, and copper have been shown (Boscolo et al. 1985; Vrzgulova 1993; Merker, Vormann, and Gunther 1996).
The potential protective effects of vitamin E towards the deleterious effects of Al in different organ systems and tissues has been of increased interest more recently: vitamin E is a potential antioxidative agent, and as it is fat-soluble, it is found in biological membranes (Rosselli et al. 1995). Vitamin E inhibits the production of reactive oxygen species (ROS), so that it effectively prevents germ-cell damage and destruction resulting from this oxidation process (Willis 1985; Chorvatovicova et al. 1991; Fraga et al. 1991; Meydani 1995; Hsu et al. 1998; Rao and Sharma 2001). Indeed, Hsu et al. (1998) reported that vitamin E prevents the mobility loss of spermatozoa by promoting a reduction in reactive oxygen species (ROS) production. This concept is thus supported by our data, where we have shown that intraperitoneal administration of Al2(SO4)3 to male rats at relatively low doses (5 mg/kg body weight; 3 times per week for 2 weeks) has toxic effects on the spermatogeneic sertoli cells and testicular tissue morphology, and that these deleterious effects can be ameliorated by coadministration of vitamin E with the Al.
For vitamin E itself, when administered alone at therapeutic levels, it does not appear to promote specific changes in living organisms. It has also been shown to have deleterious effects and to be toxic, and lethal, at levels of around 1 to 2 g/kg/day (Henry 2005). However, at the slightly lower doses used in the present study (500 mg/kg body weight), vitamin E alone showed no specific changes in testis morphology in comparison with the control samples.
As indicated above, some other antioxidative agents have also been used to abrogate Al and/or cadmium toxicity, including selenium and ascorbic acid. A lack of maternal and developmental toxicity in mice given high doses of Al hydroxide and ascorbic acid during gestation has been shown (Colomina et al. 1994), with a protective role of ascorbic acid seen towards Al-induced impairment of hematobiochemical parameters, lipid peroxidation, and enzyme activities in male rabbits (Yousef 2004).
However, our study is limited to light and ultrastructural histological changes in the testis. In addition to these effects, the monitoring of a number of the biochemical parameters following treatment with another antioxidative agent, ascorbic acid, should also provide further information relating to the system investigated in the present study. This will be addressed further in future work, along with reproductive performance tests, thus providing further information towards the elucidation of the total picture of Al2(SO4)3 and vitamin E effects on male reproductive toxicity under intraperitoneal administration.
Footnotes
Figures
The authors are grateful to “CEP” for funding the study. The authors would like to thank Dr. Chris Berrie for scientific English language editorial assistance. The authors are also indebted to Mr. Huseyin Solmaz for the processing of tissues for electron microscopy.