Effects of Cadmium and /or Chromium on reproductive organs and semen profiles of male albino rats

Address correspondence to Eman Abd Elhafeez; Tel. 01005344462; E-mail: eman1722013@gmail.com Objective: To evaluate the potential hazards of cadmium and/or chromium on the reproductive system of adult male albino rat. Design: Randomized controlled study. Animals: Forty mature male albino rats weighing 260 ± 10 g. Procedures: Rats were allocated into four groups (ten animals each). Control group (group 1), group 2 received 4.4 mg kg cadmium chloride, group 3 was given 2.5 mg kg-1 sodium dichromate and group 4 received combination of Cd (2.2 mg kg) and Cr (1.25mg kg) orally, once daily for 65 consecutive days. Results: Exposure to Cd or Cr, in particular their combination, caused a reduction in the index weights of testes, epididymis, seminal vesicle and prostate glands. They induced a reduction of sperm count and viability with an increase of abnormal sperm morphology. Interestingly, in the combination group (Cd and Cr together), the deleterious effects were more noticeable. Pathologically, both Cd and Cr produced degenerative changes in seminiferous tubules, necrosis of spermatogenic epithelium within the testis. Moreover, the interstitial tissue of epididymis showed marked edema and prostate showed necrosis and serous exudate of lining epithelium. In the interaction group, testis showed complete degenerative changes and necrosis of spermatogenic epithelium, with marked interstitial edema and hyperplastic epithelial lining of epididymal tubules. Conclusion and clinical relevance: The present results support the hypothesis that the testis is one of the most sensitive organs to Cd and/or Cr and that the exposure to any of them or to their combination lead to testicular damage and thereby male infertility.


INTRODUCTION
Cadmium (Cd) is an environmental pollutant, produced mainly during many industries such as battery, electroplating, pigment and plastic industries. The general population is exposed to Cd via pollutants found in both drinking water and food. Besides industrial sources, cigarette's smoke constitutes the most important source of Cd [1]. The International Agency for Research on Cancer (IARC) has categorized Cd as a carcinogen [2]. It has been proven that acute and chronic exposure to Cd has several detrimental impacts on many organs, especially testes in both human and animals [3].
Exposure to Cd induces infertility and cancers of the reproductive tissues [4,5]. It has been demonstrated that Cd could cause haemorrhage, edema, necrosis and atrophy of the testes. In addition, it could induce a decrease in counts and motility of sperm and reduce the concentrations of testosterone in plasma and testes [6]. Exposure to Cd could also induce a significant increase of abnormal sperm morphology [7].
Environmental Protection Agency listed chromium as one of the most environmental toxic heavy metals for human [8]. Chromium is naturally found in rocks, volcanic dust, gasses, soils as well as plant and animals. Oxidation forms of chromium, generally present in the environment, are chromium (II), chromium (III), chromium (IV), and chromium (VI) [9]. Chromium (VI) compounds are extensively used in many industries including stainless steel production, welding, electroplating, leather tanning, production of dyes, and wood preservatives [10,11]. Chromium (VI) is considered one of the most toxic transition heavy metal that has deleterious effects on the male reproductive system [12,13].
It can easily penetrate the cell, then goes through a chain reaction with production of chromium intermediates [14]. The reduced forms of Cr bind to intracellular proteins, resulting in an increase of total chromium in the blood cell. During this reduction process, chromium produces reactive oxygen species that induces oxidative stress [15]. Ingestion of hexavalent Cr compounds leads to degeneration in the outer layers of somniferous tubules, a decrease in the sperm count and spermatogonia per tubule, with subsequent increase in the percent of abnormal spermatozoa [16]. In male albino rat, hexavalent Cr induces reductions in the weights of testes, epididymis, seminal vesicles and prostate glands with a remarkable reduction in epididymal sperm number and sperm motility [17].
The combined effects of Cd and Cr on oxidative-endoplasmic reticulum-stress mediated apoptosis in the liver of mice have been investigated [18]. In a study applied on Japanese Quail eggs, the mixture of Cd and Cr decreased eggshell strength and thickness more than the individual exposure to each [19]. However, studies on the combined effects of these metals specifically on male reproductive system are scarce. Therefore, this study was delineated to evaluate the toxic effects of the exposure to Cd and Cr and their combination on the reproduction of male albino rats.

Animals
Forty healthy mature male albino rats weighing 260 ± 10 g were purchased from Animals Experimental Unit, Faculty of Veterinary Medicine, Zagazig University. They were placed in plastic cages with wood shavings as bedding, and were kept under controlled condition (23 ± 1 °C, 12 h light and 12 h dark cycle). Rats were supplied with standard laboratory pelleted feed and water ad libitum. The animals were accommodated for our laboratory conditions for 2 weeks before the start of the experiment. The animal research was accomplished in agreement with the Guiding principle for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH), and the study protocol was approved by Research Ethics Committee, Faculty of Veterinary Medicine, Mansoura University (M/23).

Chemicals
Cadmium chloride monohydrate (CdCl 2 H 2 O) and Sodium dichromate dihydrate (chromium VI) (Oxford Lab chem, Mumbai, India) were used in this experiment.

Experimental design
The experimental rats were randomly alienated into four groups (10 rats per each). (Group 1) control group was administrated 0.5 ml distilled water per rat as a vehicle, ( ). The vehicle and chemicals were given orally via stomach tube, once daily for 65 consecutive days, based on a complete spermatogenic cycle in rats [22]. At the 65 th day of the experiment, animals were exposed to light anesthesia by diethyl ether.

Determination of index weight of reproductive organs
Rats were weighted and their reproductive organs (testes, epididymis, seminal vesicles and prostate glands) were dissected out and weighted. Their index weight (I.W.) was calculated relative to the total body weight of animals. Index weight (I.W.) = [organ weight (g) / body weight (g)] x 100

Evaluation of reproductive parameters (sperm picture)
Epididymis from each rat was collected and the caudal part was gently squeezed and seminal content was obtained and diluted with 1 ml normal saline. Sperm count was performed using the Improved New Pauer hemocytometer. Determination of sperm viability and sperm abnormalities were measured by using equal amounts of semen and eosin-nigrosin stain (one drop each), which were mixed together, then a thin film was made using a clean glass slide and was examined with a light microscope at (40×). Eosin is a differential stain that is able to stain the head of dead sperms with red, while nigrosin stain is used for background staining. Live and dead sperms were expressed as a percent and sperm abnormalities were also determined [23].

Histopathological examination
Specimens from reproductive organs (testes, epididymis, seminal vesicles and prostate glands) were collected and kept in 10% neutral buffered formalin. Sections of 5-micron thickness were prepared from collected specimens, stained by hematoxylin and eosin (H&E) and examined by light microscope [24].

Statistical Analysis
Data were analyzed statistically using the statistical software program (SPSS for Windows, version 20, USA). Mean and standard errors of mean for each variable was presented. Differences between groups were assessed using one-way ANOVA with post hoc Least Significance Difference (LSD). At P < 0.05, the result was considered significant.

RESULTS
In Table 1, the index weights of reproductive organs were presented. The index weights of testes, epididymis, seminal vesicle and prostate showed significant reductions following the exposure to Cd-, Cr-and Cd + Cr compared to control with a significant reduction recorded in Cd + Cr co-exposed group compared to individual exposure.   Mans Vet Med J 20:3 (2019) [14][15][16][17][18] In each column, the means with different superscript letters are significantly different at P < 0.05. Cd refers to cadmium treated group, Cr refers to chromium treated group, Cd +Cr refers to the combination group.
In Table 2, sperm count, sperm viability and percent of sperm abnormalities revealed significant reductions in Cd-, Cr-and Cd + Cr exposed groups compared to the control group. The reduction in sperm count was more pronounced in Cd + Cr co-exposure group. Types of sperm abnormalities were bent tail, headless sperm, banana-shaped head, undulating mid piece, folded tail, coiled head, tailless head, coiled tail, detached head and curved tail (Figure 1).
Histopathological findings in different organs of rats received Cd and or Cr orally for 65 days were demonstrated in Figure 2.

DISCUSSION
The index weight of reproductive organs is considered one of the most valuable indicator for reproductive health [25]. In the present study, sub-chronic exposure to Cd or Cr significantly reduced the index weight of reproductive organs. This decrease could be resulted from the ability of Cd to cause lipid peroxidation as well as oxidative damage with subsequent atrophy in the reproductive organs [26,27]. Cadmium has the ability to cause necrotic degenerative changes in the testicular tissue leading to reduction in the testicular weight [28,29]. It has been also postulated that reduction in testicular weight associated with Cd toxicity may be due to reduction in the number of Sertoli cells and/or Leydig cells [30][31][32]. These results were confirmed by the histopathological analysis of the testes in the present study.
Likewise, Cr reduced the index weight of the reproductive organs owing to either a decrease in serum testosterone level in Cr-exposed rats [33], or the liberation of reactive radicals resulting in amplified oxidative stress damages in the sperm membranes, proteins and DNA with substantial decrease in the weight of reproductive organs [34]. It is worthy to note that a significant decrease in the index weights of reproductive organs was noticed in Cd + Cr co-exposed group, suggesting that exposure to Cd + Cr in combination is much more toxic than separate exposure to each. The reduction in the index weights of reproductive system was attributed to the oxidative stress effects induced by both compounds [34,35]. Our hypothesis is supported by the result of a study on Japanese Quail eggs, where the mixture of Cd and Cr produced more reductions in the weights of eggs more than the individual exposure to each [19]. The present result is an in agreement with that of previous results [36].
Semen analysis showed that sub chronic exposure to Cd and Cr resulted in a significant decrease in total sperm count and Mans Vet Med J 20:3 (2019) [14][15][16][17][18] viability with a significant increase in the percent of sperms with abnormal morphology. Cadmium induced damage to the testicular germinal epithelium, the Leydig cells and the Sertoli cells resulting in testicular and cellular damage which adversely affected sperm characters [7,26]. Exposure to Cd disrupted the tight junctions between Sertoli cells and altered germ cell adhesion with consequent exfoliation of immature cells into the lumen of seminiferous tubules, leading to a decrease in viable sperm count [37]. In the current study, the significant increase in sperm abnormalities could be attributed to the excessive production of oxidative stress byproducts with eventual cellular death [28,38].
Following Cr-exposure, the alteration in sperm parameters might be attributed to the impairment in steroidogenic mechanisms and hormonal derangement. In male rabbits, the alteration in sperm parameters associated with Cr-exposure has been explained as a result of decreased serum concentration of testosterone and increased serum concentration of FSH [39]. Taken together, generation of ROS and peroxidation of sperm membranes have a harmful effect on sperm motility and morphology [40].
Our results suggest that Cd and Cr together generate additive or synergistic toxic effects that lead to more pronounced decrease in sperm count, viability and an increase in sperm abnormalities compared to the separate exposure to each. Our hypothesis could be supported by the result of Skalická et al.' work on Japanese Quail eggs [19].
The histopathological findings where the testes and other reproductive organs showed marked pathological alterations could also support our hypothesis. Previous studies suggested that oxidative stress, lipid peroxidation, depletion of antioxidant defense systems and high production of pro-inflammatory mediators were involved in the pathological alterations of cadmium-induced testicular toxicity [41].
The testis is extremely sensitive to Cd toxicity, which causes profound testicular damage [29,42]. In the present investigation, the testes of Cd-treated rats exhibit degeneration, necrosis and atrophy of almost all of the seminiferous tubules [43]. Such findings coincide with that of previous reports [44,45].
Similarly, Cr could exert pathological alterations in reproductive organs in Cr-exposed rats [46]. Also, Cr caused a significant decrease in the thickness of epithelia of seminiferous tubules and this is suggested to be as a result of the degenerative changes and accumulation of sloughed cells in tubule lumen [47]. Our results agree with the results of studies conducted on male reproductive organs in rats [33,48].
Testis subjected to the combination of Cd and Cr showed complete degenerative changes and necrosis of reproductive organs. This may be due to accumulation of Cd and Cr in testis and epididymis. In a previous study in male Sprague Dawley rats, Cd and Cr combination induced organelle damage associated with the accumulation of both elements in the nuclei and mitochondria of the liver and kidney. At these sites, the metals can induce DNA-and protein damage and lipid peroxidation with subsequent functional changes in the target tissues and organs [49][50][51].

Conclusion
Co-exposure to Cd and Cr in male rats induces extra reproductive toxicity than single exposure, especially in sperm parameters. Consequently, environmental pollution with both elements may have significant clinical problems.