Effect of Vitamin E Pre-Treatment on Histopathological Changes on Rat Testis Following Cadmium Chloride Administration

 

Rekha D. Kini¹*, Tripathi Y.², C.V. Raghuveer³, Sheila R. Pai¹

¹Dept. of Physiology, Kasturba Medical College, Mangalore, Manipal University, Karnataka

²Department of Physiology, Santhosh Medical College, Ghaziabad

³Department of  Pathology, Yenepoya  Medical college, Mangalore

*Corresponding Author E-mail:- rekhadk2004@yahoo.com

 

Pollution and industrial practices result in concentration of heavy metals in the environment. Cadmium (Cd) has been recognized as one of the most toxic heavy metals and an environmental pollutant that affect the testis. From the literature it is evident that use of antioxidant in preventing cadmium chloride induced testicular damage is conflicting. So, this study was undertaken to evaluate the role of vitamin E on testicular damage induced by cadmium chloride. Rats were divided into Group I, II, III and IV. Number of rats in each group is 8. The normal control group (Gr. I) rats were administered with the single dose of normal saline intraperitoneally. Group II received Vitamin E (100mg/kg bw) orally for 30days.Group III received a single dose of 2mg/kg bw cadmium chloride and Group IV received vitamin E (100mg/kg bw) for 30 days orally prior to cadmium administration. Testes were fixed in Bouvins fluid and processed for histopathological studies. Rats in group III showed a significant decreases (p<0.001) in standard tubular diameter (462.42±33.2) and epithelial height (37.56±8.13) compared to Gr I (567.62±24.5) and II (561.76±31.6). Rats pre-treated with vitamin E (Gr.IV) showed a significant increase in STD (520.73± 45.70) and epithelial height (68.84±7.84) compared to Gr.III (p<0.001).The results showed that vitamin E showed a protective effect against cadmium chloride induced testicular damage. The results of this study may potentiate the antioxidant role of vitamin E in rescuing the testis exposed heavy metals like cadmium.

 

 

INTRODUCTION:

Environmental reproductive health focuses on exposures to environmental contaminants (synthetic chemicals and metals) and their potential effects on all aspects of reproductive health. Humans are exposed daily to a mixture of environmental contaminants in air, water and food. Cadmium is an extremely toxic metal and an environmental pollutant. The increasing use of cadmium in industry also increases the risk of human exposure to this metal^1,2. Exposure may be either occupational (mainly by inhalation) or nonoccupational (by cadmium contamination of drinking water or food). It is also widely used in mining, metallurgical operations, electroplating industries manufacturing vinyl plastics which used in metallic and plastic pipes. Cadmium is known to exert toxic effects on multiple organs, including the testes. It causes tissue damage in humans and animals, acting as an inducer of oxidative stress^3,4 and an endocrine disruptor in humans and rodents.

 

Studies have illustrated that the testis is exceedingly sensitive to cadmium toxicity⁵, associated with reduced male fertility, such as reduced spermatozoa count and poor semen quality, in men exposed to cadmium and other environmental toxicants⁶.Several lines of studies indicate that ROS are involved in cadmium chloride induced testicular damage⁷. Antioxidants protect sperms from damage caused by reactive oxygen species (ROS), and oxygen radical scavengers have preventive effects on testicular function after cadmium toxicity, thus suggesting that antioxidants act as a factor for survival and ensure germ cell function⁸.Alpha (α)-tocopherol (vitamin E) is one of eight forms of vitamin E⁹. The cell membranes and plasma lipoproteins contain α-tocopherol, a lipid-soluble molecule that functions as a chain-breaking antioxidant. Vitamin E is widely available in the market in the oral form. Experiments on laboratory animals have shown that increased intake of vitamin E leads to the generation of fewer toxic free radicals. Few studies have been conducted on cadmium toxicity using vitamin E as the antioxidant¹⁰. Therefore, the present study was designed to address the issue of cadmium induced testicular toxicity and to examine the role of vitamin E as an antioxidant in ameliorating the testicular damage induced by cadmium chloride in male Albino rats.

 

MATERIAL AND METHODS:

The present study was conducted following approval from Institutional Bioethical Committee and strict internationally accepted guidelines, for the usage of animals in experimental study were followed. Inbred adult male albino rats of wistar strain weighing 200-250g were used in the present study. Animals were housed in polypropylene cages (4-5 rats per cage) under standard laboratory conditions and fed ad libitum with commercial rodent chow (Hindusthan Lever Limited) and water. Cadmium chloride (CdCl₂) (Loba Chemie, India) was dissolved in normal saline (0.9%). Vitamin E was dissolved in oil and administered intraperitoneally.

 

Experimental protocol and drugs:

Animals were divided into four groups of eight rats in each group. The normal control group (Gr. I) rats were administered with the single dose of normal saline intraperitoneally. Group II received Vitamin E(100mg/kg bw) orally for 30days.In Group III  (cadmium treated group) rats were administered with single dose2mg/kg bw  cadmium chloride intraperitoneally. In pretreated groups, rats were pretreated with vitamin E (100 mg/kg bw) for 30 days orally prior to cadmium administration (2mg/kg bw). In all the groups, rats were sacrificed under anesthesia 15 days after the final cadmium administration. Following the completion of the experimental protocol animals in each group were anaesthetized by injecting sodium pentabaritone (40mg/kg bw) intraperitoneally under aseptic conditions. Laparotomy was performed and the reproductive organs were exposed. Both the testes were removed and cleaned of fat tissue and blood and weighed. To obtain the quantitative analysis of testicular damage, the tissues were processed and paraffin blocks were prepared as per standard protocols¹¹. Five-micron-thick sections were obtained and stained with haematoxylin and eosin for light microscopic analysis. To measure the standard tubular diameter (STD), five transversely cut seminiferous tubules from each testis were selected randomly and measured per cross-section using a stage micrometer that was calibrated with an eyepiece micrometer. The two diameters of the tubules, one perpendicular to the other, were measured. The average of the transverse and perpendicular diameters was taken for each animal¹².To measure the standard epithelial height (SEH), five transversely cut seminiferous tubules were randomly selected and measured per cross-section tubules according to Canan et al’s method¹².

 

 

For qualitative evaluation, the testes were examined for the presence of coagulation-type necrosis and classified into four grades¹³. Grade 0 showed an absence of coagulation and necrosis in the seminiferous tubules. Grade 1 indicated slight coagulation, with < 25% of the seminiferous tubules containing evidence of necrosis. Grade 2 indicated moderate coagulation with ≥ 25% of the seminiferous tubules containing variable degrees of necrosis, Grade 3 indicated severe damage where ≥ 75% of the seminiferous tubules demonstrated complete necrosis.

 

Statistical analysis:

Values were expressed in mean ± SEM. SPSS version II was used for statistical analysis. Differences between groups were assessed by one-way analysis of variance .The Post Hoc (LSD) test was used for intergroup comparison. P <0.05 was taken as significant.

 

RESULTS:

Rats administered with 2mg/kg bw cadmium chloride (Gr.III) showed a significant decrease in the STD and SEH compared to normal control (Gr. I) as well as vitamin treated group (Gr.II). However, rats that were pretreated with vitamin E (Group IV) showed significant improvement in these parameters when compared to the untreated control group (Group III).Results of the histopathological analysis of the various control and test groups are summarized in Table II. The normal architecture of the seminiferous tubules for Group I and II is shown in Figs. 1 and 2, respectively. The rats that were administered with Cadmium chloride (Group III) showed severe destruction of the tubular cells, i.e. Grade 3 (Figs. 3).The majority of the cells showed degeneration, especially the spermatocytes and spermatids and the tubules had hardly any sperms when compared to Group I and Group II. The haematoxylin and eosin-stained section of Group IV rats, the vitamin E pre-treated group (Fig.4), showed lesser atrophic and degenerative changes (Grade 1) to the tubular epithelium when compared to the untreated control group (Group III). The seminiferous tubules in many of the sections showed near-normal architecture.

 

 

 

 

Table I: Effect of vitamin E pretreatment on cadmium induced testicular toxicity on STD and SEH. Number of animals in each group is 8. Values are expressed as Mean± SD.P<0.05 taken as significant.

Group	STD(µm)	SEH(µm)
I	567.62±24.5	71.16±4.36
II	561.76±31.6NA	73.81±3.87NA
III	462.42±33.2§	37.56±8.13§
IV	520.73± 45.70¶	68.84±7.84¶

^NAGroup II vs. Group I: p-value is not significant

§Group III vs. Group I and II: p-value < 0.001

¶ Group IV vs. Group III: p-value < 0.001

SD: standard deviation; STD: standard tubular diameter;

SEH: standard epithelial height

 

 

Table II:Effect of vitamin E pre-treatment following cadmium chloride administration on histopathological changes in rat testis

Group	Grade
I	Grade 0	Normal testicular architecture with an orderly arrangement of germinal cells. Absence of coagulation and necrosis in the seminiferous tubules
II	Grade 0	Normal testicular architecture with an orderly arrangement of germinal cells. Absence of coagulation and necrosis in the seminiferous tubules
III	Grade 3	Seminiferous, with  necrosis of germinal cells. 75% or more of the seminiferous tubules demonstrated complete necrosis.
IV	Grade I	slight coagulation, with < 25% of the seminiferous tubules containing evidence of necrosis

Fig. 1 Photomicrograph of rat testis shows normal histological features in the normal control group (Group I) (Haematoxylin and eosin, ×10)

 

Fig. 2 Photomicrograph of rat testis shows normal histological features in the vitamin E treated group (Group II) (Haematoxylin and eosin, × 10).

 

Fig. 3 Photomicrograph of rat testis showing necrosis in more than 75% of the tubules in cadmium administered rats (Group III) (Haematoxylin and eosin, × 10).

 

Fig. 4Photomicrograph shows less than 25% of necrosis in the seminiferous tubules of rats that were pre-treated with vitamin E prior to the cadmium chloride administration (Group IV)(Haematoxylin and eosin, × 10)

 

DISCUSSION:

In the field of toxicology, the adverse effects of greatest concern are those of chronic toxicity, cancer and reproductive dysfunction¹⁴. Cadmium is a potent human carcinogen and occupational exposure to it has been associated with cancers of lung, the prostate and the kidneys¹⁵. The production of ROS is greatly enhanced under the influence of various environmental and lifestyle factors such as pollution and smoking¹⁶. But, excess production of free radicals or reactive oxygen species (ROS) can damage sperm and ROS have been extensively studied as one of the mechanisms of male infertility¹⁷. Aruld has reported that oxidative stress by free radical toxicity caused by cadmium affected infertility¹⁸.Therefore, to maintain viable reproductive ability; a protective mechanism against ROS is of importance. This function is taken up by the antioxidants present in the body. The results of the present study showed a significant decrease in STD and SEH as well as complete destruction of testis. Studies have shown that antioxidants play a critical role in monitoring sperm dysfunction and infertility. Blanco et al. claimed that even with low doses of cadmium chloride (1mg/kg for one month) induced lack of spermatogenesis and severe necrosis of the testes of rats ¹⁹. Moreover, Santos et al. reported that endothelial damage of the small blood vessels, edema and hemorrhage of the rat testes can be demonstrated by using just a single parenteral dose of cadmium chloride at 2-4 mg/kg²⁰. In the present study, pre-treatment of rats with vitamin E showed a decrease in the testicular tissue damage induced by cadmium. In the present study, the rats that were treated with vitamin E showed an increased the STD and SEH compared to the untreated experimental controls. However, upon pre-treatment with antioxidant vitamin E (Group IV), the seminiferous tubules in these rats showed less damage when compared to the untreated control group.

 

The results of the present study also show that vitamin E plays a significant role in reducing histological damage induced by cadmium chloride administration. Studies have shown that vitamins C and E are essential for normal spermatogenesis and that in the absence of such vitamins, the animals showed dysfunction of the germinal layers²¹. Supplementation of vitamins C and E has been found to reduce testicular ROS and restore normal testicular function in cadmium-exposed rats²². Both these vitamins have often been simultaneously used in clinical and experimental medicine, as they synergistically exerta potent antioxidant action, which can provide protection against excessive oxidative damage. Therefore, the results of the present study suggest that vitamin E, when administered before cadmium administration offers significant protection against acute testicular damage in rats.

 

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Accepted on 10.06.2013      © RJPT All right reserved