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Changes in semen characteristics and sex hormones of rats treated with iron oxide nanoparticles, silver nanoparticles and their mixture

Few studies have demonstrated the toxicity of iron oxide nanoparticles (Fe2O3NPs), and silver nanoparticles (AgNPs), and their combination on reproductive performance.Therefore, the present study aimed to investigate the reproductive toxicity of iron oxide nanoparticles, silver nanoparticles and their combination in male rats. Forty Wistar male rats were divided into 4 equal groups. Group 1 served as control, group 2 was administered orally with Fe2O3NPs (5 mg/kg BW; >50 nm), group 3 was treated intraperitoneally with AgNPs (50 mg/kg BW; >100 nm) and group 4 was administered with the mixture of Fe2O3NPs with AgNPs. Animals were treated with doses every day for 79 days. The results revealed that semen parameters (sperm count, abnormal sperm and sperm motility) of adult male rats treated with Fe2O3NPs, AgNPs and their combination showed significant (P<0.05) reduction in sperm motility and sperm count, and significant (P<0.05) induction in abnormal sperm compared to the control group. Results also showed significant (P<0.05) decrease in the levels of testosterone and TSH, and significant (P<0.05) increase in FSH, LH, T3 and T4 levels in rats treated with Fe2O3NPs, AgNPs and their combination compared to the control group. The specific activity of testicular 17β-hydroxysteroid dehydrogenase (17β-HSD) was significantly decreased, while 17-ketosteroid reductase (17-KSR) was significantly increased in animals treated with Fe2O3NPs, AgNPs and their combination compared to the control group. It was concluded that rats exposed to Fe2O3NPs, AgNPs or their combination caused testes inflammation and sex hormones imbalance that affect reproductive performance.


Introduction
Iron oxide nanoparticles, subsequently increasing their existing levels in the environment and human exposure, the increased exposure can influence upon society in many ways, such as potential risk to human health, in particular to pregnant women or babies. Therefore, it is critically important to understand the developmental and reproductive toxicity and the long-term effects of iron oxide nanoparticles [1]. Nowadays, an important growing in nanotechnology has lead to the increased production and application of several nanostructure materials, including silver nanoparticles (AgNPs). This kind of nanoparticles has been incorporated into several products such as cosmetics, textiles, and medicines for their bactericidal effect [2,3]. Because of their antimicrobial, optical, and catalytic properties, silver nanoparticles (SNP) have gained particular interest for many commercial applications. According to the Woodrow Wilson Inventory, approximately 30% of all nanoparticles-enabled products contain nanosilver. Thus, SNP are highly commercialized and are now being used in many daily life products mainly because of their antimicrobial properties. In clear contrast to SNP widespread use, putative health effects have only just begun of being adequately addressed [4].Very little is known about the toxicity of nano-sized silver particles, however, the size and surface area are recognized as important determinants for toxicity [5]. Significant concerns have been expressed about the potential risk of silver nanoparticles (AgNPs), due to the current and projected high exposure [6]. A few research groups have investigated the toxicity of silver nanocomposites and nanoparticles in cell lines to estimate viability and reactive oxygen species (ROS) generation [7 -9].Despite the wide application of nanoparticles, there is a serious lack of information concerning their impact on human health and the environment [5]. From the previous literature, there is a shortage in the information on the effects of iron oxide nanoparticles (Fe2O3NPs), silver nanoparticles (AgNPs) or their combination on male fertility and their mechanism of reproductive toxicity.

Aim of the study
The present study was carried out to investigate the reproductive toxicity of Fe2O3NPs and AgNPs alone or in combination in male rats fertility parameters (semen characteristics, sex hormones, testosterone, luteinizing hormone, follicle-stimulating hormone, thyroid stimulating hormone, thyroxine and triiodothyronine).

Material and methods
The epididymis was sampled at the end of the experiment. Spermatozoa obtained from the caudaepididymis were subsequently diluted with physiologicalsolution (20 μl) at 37 o C. The sample was located in the Makler chamber (Sefi-Medical Instruments, Germany). Analysis was realized using a CASA System-Supervision (Minitüb, Tiefenbach, Germany) with Olympus BX 51(Olympus, Japan) microscope as described by Krause 10]. The sperm count; motile sperm percentage and abnormal spermmorphology (%) were evaluated in the experimental groups according to the method of Dunson et al. [11]. A drop of 1% eosin-Y staining was added to the sperm sample on amicroscope slide at 400x magnification for morphological studies [12].Plasma testosterone rat enzyme-linked immunosorbent assay (ELISA) is a competitive immunoassay for the quantitative measurement of testosterone in rat, was estimated by the method of Zirkin and Chen [13] and Sakuma [14].The ELISA technique uses antibodies with high affinity and specificity for two different epitopes on rat follicle stimulating hormone (FSH) was estimated by the method of Kjeld et al. [15]. Furthermore, the ELISA kit applies to the in vitro quantitative determination of rat luteinizing hormone (LH) concentrations in plasma was estimated by the method of Closset and Hennen [16] and Teerds et al. [17]. Rat thyroid stimulating hormone (TSH) was assayed by using ELISA kit for the quantitative determination of rat TSH concentrations in plasma (CUSABIO BIOTECH CO., LTD.). Triiodothyronine (T3) was assayed by using ELISA Kit for the quantitative measurement of (T3) in rat plasma (Abnova CO., KA 0925). Moreover, (T4) was assayed by using ELISA kit in vitro quantitative detection of rat in plasma (Biovision CO., U.S.A). Streoidgenic 17-ketosteroidreductase enzyme activity (17-KSR; EC 1.1.1.64) was estimated by the method of Katryna and Anita [18]. Specific activity of 17β-hydroxyl steroid hdrogenase enzyme activity (17β-HSD; EC 1.1.1.51) was estimated by the method of Bogovich and Payne [19].Interleukin-6 (IL-6) ELISA Kit for the in vitro quantitative measurement of rat IL-6 in tissue homogenates was estimated by the method of Ferguson-Smith et al. (1988). Tumor Necrosis factor alpha (TNF-α) rat in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of rat TNF-α in cell culture supernatants. It was estimated by the method ofHedayati et al. [20].The tumor suppressor P53 acts primarily as a transcriptional activator that controls the expression of many genes was estimated by the method ofHupp et al. [21], Renard et al. [22], Gottifredi and Prives [23] and Yang et al. [24]. The Trans AM Kit combines a fast and user-friendly ELISA format with a sensitive and specific assay for transcription factors. TransAM p53 Kits contain a 96-well plate on which has been immobilized an oligonucleotide that contains a p53 consensus binding site (5´-GGACATGCCCGGGCATGTCC-3´). P53 contained in nuclear extract specifically binds to this oligonucleotide. The primary antibody used in TransAM p53 Kit recognizes an accessible epitope on p53 protein upon DNA binding. Addition of a secondary HRP-conjugated antibody provides a sensitive colorimetric readout easily quantified by spectrophotometry.

Results and discussion
The changes in semen characteristics (sperm count, sperm motility and abnormal sperm) of adult male rats treated daily for 79 days with iron oxide nanoparticles (Fe2O3NPs), silver nanoparticles (AgNPs) and their combination are summarized in Table 1 and Figures 1. Results showed that Fe2O3NPs, AgNPs and their combination caused significant (P<0.05) reduction in sperm motility and sperm count, and significant (P<0.05) induction in abnormal sperm compared to the control group. The effects of the combination of Fe2O3NPs and AgNPs were more toxic than each one. Table 2 and Figures 2 and 3 represented data of testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroidstimulating hormone (TSH), Thyroxin (T4) and tri-iodothyronine (T3) of adult male rats treated with Fe2O3NPs, AgNPs and their combination. Results alsoshowed significant (P<0.05) decrease in the levels of testosterone and TSH, and significant (P<0.05) increase in FSH, LH, T3 and T4 levels in rats treated with Fe2O3NPs, AgNPs and their combination compared to the control group, and these effects were more pronounced in the combination group. Mean values within a row not sharing a common superscript letters (a, b, c) were significantly different, p < 0.05.

Figure 1Mean
values ± SE of semen characteristics and hormone levels of male rats treated with iron oxide nanoparticles (Fe2O3NPs),silver nanoparticles (AgNPs) and their combination The reproductive and developmental toxicity of nanomaterials has become increasingly recognized as an important part of the nanotoxicology (1). Yoshida et al. [25] found that nanoparticles produced during the process of diesel engine fuel combustion caused reduction in the fertility. They reported that the reduction is related to Leydig cell degeneration and the damage to the seminiferous tubules. Also, the presence of cytokines disrupts testosterone synthesis process in leydig cells by directly affecting the expression of the genes which are influential on testosterone process and the following inhibition of testosterone synthesis. So, cytokines presence is deemed to be a chronic disruptive factor for cholesterol synthesis [26].Nano-particles can cause inflammation of the epididymis, which has a role in reduction of sperm motility [27]. Carlson et al. [28] reported that three factors combine to render sperm particularly susceptible to free radical damage, a high membrane concentration of polyunsaturated fatty acids, active generation of free radicals and a lack of defensive enzymes. All of these factors combine to make the health of the sperm critically dependent upon antioxidants. Yoshida et al. [29] reported that nanoparticles increase free radicals in the sperm cells. This can damage the sperm membrane and flagellum structure and disrupt sperm motility and morphology. Free radicals lead to peroxidation of phospholipids in the mitochondria of the spermatozoa and thus impair their ultimate motility [30].
It is also demonstrated that reactive oxygen species (ROS) level is positively correlated with the proportion of sperm with amorphous heads, damaged acrosomes, midpiece defects, cytoplasmic droplets and tail defects [31]. Nasri et al. [32] found that Fe2O3 nanoparticles cause a meaningful reduction in sperm numbers and mobility and cause the inflammation of epididymis and increasing of free radicals. In accordance to these literatures, the present study showed that treatment with Fe2O3NPs, AgNPs and their combination caused significant increase in testes thiobarbituric acid reactive substances (TBARS), nitric oxide (NO), and decrease in the activities of antioxidant enzymes (SOD, CAT, GPX and GST), total antioxidant capacity and reduced glutathione in testes and this may be caused deterioration in semen characteristics (Table 1 and Figure 1). The decrease in sperm motility and sperm count is in agreement with Nasri et al. [32]who studied the effects of iron oxide nanoparticle (5, 10, 20 and 40 mg/kg BW) on sperm numbers and motility in male mice. The investigation of sperm mobility in the epydidimal area shows that the percentage of sperm mobility in the mice in experimental group in relation to control group has had a meaningful decrease, that it can be said the reason is related to the effect of iron oxide nanoparticles on mitochondrial action [7]. The researches which have done on the effect of nanoparticles on mitochondrial action in different cells have shown the reduction of mitochondrial action in all cases. On the line, 4-18 cells showed that nanoparticles such as iron oxide and silver can cross the membrane of sperm and they can connect to mitochondria and acrosome of sperm cases [7]. On the other hand, sperm mobility was happened in epididymis, the investigation shows that nanoparticles can effect on epididymis and cause the inflammation of epididymis that it can reduce sperm mobility. Also nanoparticles in cells cause the increasing of ROS that this can decrease the mobility of the cells by damaging to the flagella structure of the sperms [29]. Metal nanoparticles increase ROS such as super oxide which may cause the inhibit of RNA polymerase [29], the oxidation of molecules such as protein and even DNA and in this way they cause the decrease of LH, testosterone and Leydig cells [33]. Table 2 Mean values ± SE of testosterone, follicle stimulating hormone, luteinizing hormone, thyroid-Stimulating, hormone, tri-iodothyronine and thyroxin of male rats treated with iron oxide nanoparticles (Fe2O3NPs), silver nanoparticles (AgNPs) and their combination.

Control Fe2O3NPs AgNPs Fe2O3NPs+AgNPs
Testosterone (ng/ml) 6. The present study showed that treatment with Fe2O3NPs, AgNPs and their combination caused significant increase in T4 and T3 (Table 2 and Figure 3). In rats, T3 affects testis maturation, and thyroid receptor (TR) type-1 (TR-1) expression in rats' testes [34,35]. Maximal Sertoli cell proliferation coincides with maximal T3 binding capacity in testis, suggesting that the main target of T3 action is the Sertoli cell. However, T3 also plays a significant role in differentiation of the seminiferous epithelium, and studies in rodents have shown that T3 is an important factor in maturation of Leydig cells. The presence of T3 is necessary to initiate differentiation of mesenchymal cells into Leydig progenitor cells, and T3 works in concert with other hormones (luteinizing hormone; LH and Insulin-like growth factor 1; IGF-1) to promote Leydig cell development [36].
It was concluded that rats exposed to Fe2O3NPs and AgNPs and their combination caused testes inflammation and sex hormones imbalance that affect semen characteristics.

Figure 2
Mean values ± SE of testosterone, follicle stimulating hormone and luteinizing hormone of male rats treated with iron oxide nanoparticles (Fe2O3NPs),silver nanoparticles (AgNPs) and their combination. Table 3 and Figure 4 showed steroidogenic enzymes (17β-hydroxysteroid dehydrogenase; 17β-HSD and 17-ketosteroid reductase; 17-KSR) of adult male rats treated daily for 79 days with iron oxide nanoparticles (Fe2O3NPs), silver nanoparticles (AgNPs) and their combination. Data showed that Fe2O3NPs, AgNPs and their combination caused significant (P<0.05) increase in 17β-HSD activity and significant (P<0.05) decrease in 17-KSR comparison with the control group, and these effects were more pronounced in the combination group than each one. The increase in testicular 17β-hydroxysteroid dehydrogenase (17-β HSD) and decrease in 17-ketostroid reductase (17-KSR) may lead to the decrease of testosterone. One of the indicators of the chemical toxicity on reproductive system is the decreased level testosterone [37]. The decline in the activity of 17-KSR in animals treated with IONPs and AgNPs indicates their adverse effect on the production of testosterone hormone which in turn effects on the process of fertility via decreasing testosterone hormone production and sperm production and proliferation and hence motility, count and abnormality. On the other hand, the activity of 17β-HSD that convert testosterone to androstenedione, significantly increased in rats treated with AuNPs compared to control group.

Figure 3
Mean values ± SE of thyroid-Stimulating hormone, tri-Iodothyronine and thyroxin of male rats treated with iron oxide nanoparticles (Fe2O3NPs),silver nanoparticles (AgNPs) and their combination.

Conclusion
It was concluded that rats exposed to Fe2O3NPs, AgNPs or their combination caused testes inflammation and sex hormones imbalance, which may affect reproductive performance. The result of this study provided insight into the importance between reproductive performance and toxicity of nanoparticles, which are widely incorporated in cosmetics, textiles, and various biomedical applications. Investigations should be considered carefully due to their impact upon society in many ways, such as potential risk to human health.