Toxicity Investigation of Nano-SiO2 on Male Reproductive System in Pubertal Mice

Background Puberty is a crucial stage to gain reproductive capacity, but it is also a period vulnerable to exogenous materials. While exposure to nanoparticles (NPs) has been linked to toxic responses in reproductive system in previous ndings, little is known about the age-dependent effect of NPs, let alone the underlying mechanism. In the present study, we assessed male fertility parameters and explored its mechanism following intraperitoneal exposure to Nano-Silicon dioxide (Nano-SiO 2 ) in mice during puberty.


Abstract Background
Puberty is a crucial stage to gain reproductive capacity, but it is also a period vulnerable to exogenous materials. While exposure to nanoparticles (NPs) has been linked to toxic responses in reproductive system in previous ndings, little is known about the age-dependent effect of NPs, let alone the underlying mechanism. In the present study, we assessed male fertility parameters and explored its mechanism following intraperitoneal exposure to Nano-Silicon dioxide (Nano-SiO 2 ) in mice during puberty.
Methods 40 mice aged 5 weeks were divided into 2 groups after 1 week acclimation and then exposed to 40mg/kg Nano-SiO 2 dissolved in saline or vehicle controls by intraperitoneal injection every day over a period of 7day, respectively. Changes in the structure and function of male reproductive organs were detected after exposure.

Results
Nano-SiO 2 exposed through intraperitoneal injection could cause damage to the testicular and epididymal histological architecture and reduce the level of sex hormone (testosterone), leading to a decrease in sperm quality and quantity. Furthermore, Nano-SiO 2 could induce oxidative stress and in ammation in male reproductive tissues, indicated by reduced activity of antioxidants (superoxide dismutase, SOD) and increased level of the lipid peroxidation marker (malondialdehyde, MDA), which leads to the activation of cell apoptosis.

Conclusion
Exposure to Nano-SiO 2 in pubertal mice could cause toxicity on male reproductive system via inducing oxidative stress and activating TNF-α mediated apoptotic pathway.

Background
During the past forty years, males in multiple countries all over the world have experienced a decline of more than 50% in the quantity and quality of semen which partially responsible for the 10-15% prevalence of infertility [1,2]. Meanwhile, a spectacular rise in testicular cancer and other male reproductive diseases, including disorders of spermatogenesis, have occurred in all parts of the world [3]. The high incidence of male reproductive disease may be caused by the widespread of environmental pollution and the sensitivity of male reproductive system to exogenous hazards, especially during crucial windows of development.
Puberty is the period of transition between childhood and adulthood, characterised by the development of gonadal maturation and attainment of reproductive capacity [4]. As for male, puberty is thought to occur approximately from postnatal day [PND] 42 to 49 in rodents and the completion of rst wave of spermatogenesis is considered as the onset of puberty [5]. In male mice, from day 7 to 35 postpartum (pp), spermatogonia within the seminiferous tubules transform through the differentiated spermatogenic cells to become spermatozoa [6]. The rst round of spermatogenesis is followed by additional waves to ensure continuous sperm production throughout the life of the animal. It is worth noting that the starting and running maintenance of spermatogenesis are under the control of hypothalamus-pituitary-gonadal (HPG) axis, one branches of the endocrine system essential for the integrity of male reproductive barrier, which is gradually being established during puberty [5]. Thus, disruptions by various stressors during puberty could have long-term effects on male fertility.
In the last two decades, the vigorous development and broad application of nanotechnology have increased the risk of human exposure to nanoparticles (NPs) and raised concerns about their biological effects. As one of the top ve commonly used NPs in the nanotechnology consumer products, silica nanoparticles (SiNPs) have been found in almost all aspects of life, ranging from consumer goods to medicine, due to its unique physicochemical properties such as high levels of electrical conductivity, tissue permeability and resistance to biodegradation in the cellular environment [7][8][9]. However, the novel characteristics that differs from that of the bulk material has attracted global attention regarding the safety and potential adverse effects of SiNPs [10]. Indeed, studies have shown that NPs could have toxic effects on many organs including brain, liver and lungs, which are the worst affected target organs.
Reproductive toxicity is increasingly becoming considered as an important part of overall toxicology of NPs since reproductive system are believed to be essential for transmitting genetic information to the offspring and sensitive to exogenous hazards. It is reported that exposure generally to SiNPs could affect the development of testis, perturb the production of sex hormones and disturb spermatogenesis and sperm maturation which leading to reduced male fertility [11][12][13]. There is evidence that those observations could be attributed to oxidative stress toxicity, for scavenging the ROS via antioxidants (Nacetyl-L-cysteine, NAC) can ameliorate the toxicity induced by NPs [14][15][16]. Those nding have advanced our understanding of how male fertility is affected by NPs, however, the molecular mechanism underlying is still unclear. Moreover, the previous studies mainly focused on the adult stage of administration in nanoparticles-induced male reproductive damage, which fail to see the age-dependent effect of NPs [13,17]. It is reported that testis at puberty showed a higher sensitivity to exogenous contaminants, exposure to stress during this period might cause diverse outcomes that differs from that are sexually mature [18]. Therefore, it is meaningful to identify the toxic factors on male fertility during puberty and underlying their mechanisms of action to prevent their lasting effects.
In this study, we investigate the adverse effect of Nano-SiO 2 on male reproductive system in mice during puberty, including the histopathological changes in testis and epididymis, and the potential mechanism of toxicity. These ndings could provide a scienti c basis for evaluation the risk of Nano-SiO 2 to reproductive health and support the application of nanotechnologies by minimizing the adverse effects of nanoparticles in vulnerable populations.

Characteristic of Nano-SiO 2
Despite the fact that the physicochemical of silica nanoparticles commercially obtained has been preliminary evaluation, it's necessary to con rm the indicated features [19]. The morphology of 10-20nm Nano-SiO 2 in distilled water was visualized by TEM and is presented in Fig. 1A. Nano-SiO 2 showed irregularly shaped and tended to form agglomerates. TEM observation was in agreement with the results from Zetasizer measurement (Fig. 1B). The Hydrodynamic size and zeta potential of Nano-SiO 2 were 439.6nm and -27.3mV respectively (Fig. 1C), suggesting that Nano-SiO 2 in water was prone to form aggregates, which in line with nding from Magdalena Kusaczuk et al [20].
Reproductive organ coe cients are changed by Nano-SiO 2 exposure It is believed that an organ coe cient is a key biological characteristic for evaluating the function of experimental animals [21]. To determine the general toxicity of Nano-SiO 2 in mice, the weight of body was monitored daily during whole experimental period and the results showed that no changes were detected compared to controls. As for the in uence of Nano-SiO 2 on reproduction, the weights of reproductive organs including testis and epididymis were dissected and measured the day after drug withdrawal. Interestingly, it turned out that the coe cient of testis is signi cantly decreased (p<0.05) while the epididymis index signi cantly (p<0.05) increased post exposure (Fig. 2).
Adverse effects of Nano-SiO 2 on testicular and epididymal pathology To determine the effect of Nano-SiO 2 on reproductive toxicity, we examined the H&E stained cross sections of testis and epididymis under light microscope to assess the pathological changes. The result showed that the cells in seminiferous epithelium, including spermatogenic cells at different stages of division and Sertoli cells, were regularly arranged in control mice (Fig. 3B). In contrast, vacuolization of seminiferous tubules (Fig. 3B) and decreased in the thickness of the seminiferous epithelium were observed in testicular sections (Table S1). Moreover, hemorrhage was detected in the interstitial tissues surrounding Leydig cells of the testicular section (white arrow in Fig. 3B) from exposed mice.
Swellings were present on the cauda epididymis in all Nano-SiO 2 treated mice (Fig. 4A), which is consistent with the increased index of epididymis (Fig. 2C). In the initial and caput segment of epididymal tubules in exposed mice, principal cells revealed similar features to that of control group, while the microvilli in the free side of the principal cells were nearly undetectable in the corpus segment (Fig. 4B). The cauda epididymis was the most seriously affected regions in exposed mice compared to controls, the principal cells were shrunken and vacuolated, and some even shedding of epithelium into the lumen (Fig.  4B). What's more, the basement membrane between epithelium and bers disappeared in mice post injection, suggesting the reproductive barrier in epididymis might be corrupted.

Nano-SiO 2 perturbs spermatogenesis and secretion of testosterone
Spermatogenesis is a complex process occurred in seminiferous tubules which give rise to highly differentiated spermatid. And the outcomes of this process, including daily sperm production (DSP) and sperm quality, are believed to be key indicators of male reproduction. To further determine the effect of Nano-SiO 2 on male fertility, we analyzed the DSP and sperm quality in mice. The results showed that the DSP and sperm count in the exposed group were signi cantly lower than that in saline group (Fig. 5A). Moreover, different malformed sperm including looped tail, bent tail, curved tail and detached head sperm were observed in exposure mice, which result in the rates of abnormal increased signi cantly (Fig. 5B, C). The process of spermatogenesis is subjected to the neuroendocrine hypothalamic-pituitary-gonadal (HPG) axis together with local testicular steroids [22]. To reveal the underlying mechanism, we examined the level of the most important androgen, testosterone, in serum. The results showed that the concentration of testosterone markedly decreased in the exposed group (Fig. 6A). What's more, compared with the control group, the expressions of androgen receptor and genes regulating androgen biosynthesis (luteinizing hormone/ choriogonadotropin receptor, steroidogenic acute regulatory protein) also declined after exposure (Fig. 6B).

Nano-SiO 2 aggravates oxidative stress and in ammation response
To get a closer insight into cytotoxicity induced by Nano-SiO 2 , the sensitive indices of oxidative stress response were determined by using SOD (superoxide dismutase) and MDA (malondialdehyde) kit.
According to the instructions of the protocol, the supernatants were added to the reagents for measuring the activity of SOD or the concentration of MDA. The results showed that the levels of MDA in treated group were markedly increased compared with control and the activity of SOD also changed after exposure (Fig. 7A). Interestingly, the variation trend in epididymis is opposite to those in testis and inconsistent with some reports nding inhibition of SOD activity once oxidative stress occurred ( Fig. 7B) [23]. It could be explained that the elevated activity of antioxidant enzymes might be a response towards Nano-SiO 2 , while the decreased activities of antioxidant enzymes could be a result of the overconsumption by ROS. The protein concentration was determined by BCA assay.
It is reported that oxidative stress is linked to in ammation reciprocally [24]. To further explore the effect of Nano-SiO 2 on reproductive organs at the molecular level, expressions of key in ammatory cytokines were detected. Results demonstrated that the mRNA expression of TNF-α and IL-1β were signi cantly upregulated after exposure, indicating Nano-SiO 2 treatment produced a robust in ammation response in testis and epididymis ( Fig. 8) Nano-SiO 2 induces DNA damage in testis and epididymis which leading to cell apoptosis It has been found that continuous oxidative stress could cause severe damage to cellular macromolecules such as proteins, lipids and DNA, resulting in cell apoptosis. To understand the concrete mechanism of Nano-SiO 2 -induced reproductive toxicity, apoptotic cells from testis and epididymal sections were detected by TUNEL staining. As shown in Fig. 9, numerous apoptotic cells were observed in the mice post exposure. Interestingly, the apoptotic sensitive cells in testis were mainly in spermatogenic cells while in epididymis they are both in spermatogenic cells and somatic cells. These data gured out the adverse effect of Nano-SiO 2 on male reproductive tissues through activating apoptotic programmed death.
TNF-α mediated apoptotic signaling pathway is activated by Nano-SiO 2 Given that TNF-α is a biomarker to in ammation induced by NPs and it is considered the master regulator of one of the death-receptor mediated apoptosis pathways, we investigate the expression of proteins involved in TNF-α signaling pathway by western blot. Our results showed that the expression of proapoptotic factors including caspase-8 and caspase-3 were upregulated post exposure. Interestingly, the expressions of anti-apoptotic factors, such as bcl-2, also increased after treatment (Fig. 10).

Discussion
Environment pollutions and lifestyle modi cation are believed to be the main cause to the high prevalence of reproductive disorders in recent years [25]. Large-scale production and utilization of NPs have increased the risk of human exposure [26]. Indeed, it has been demonstrated that the accumulation of NPs could cause damage to the structure of reproductive organs in adult model organisms [27]. However, whether there is an age-dependent toxicity of NPs remains to be further studied. Puberty is a crucial stage of maturation of reproductive system while also a period vulnerable to certain environmental factors. Exposure to stressors during or around this period is believed to have enduring consequences on multiple aspects of organism's health [5]. In this study, we aim to evaluate the adverse effect of Nano-SiO 2 on male reproductive organisms in pubertal mice and explore their underlying mechanism.
It is believed that an organ coe cient is a key biological characteristic for evaluating the function of experimental animals [21]. In this study, we found that the administration of Nano-SiO 2 did not affect the body weight of the mice but caused a signi cant reduction in the testis coe cient ( Fig. 2A-B). The weight of testis is largely dependent on the seminiferous tubules which composed of Sertoli cell and differentiated spermatogenic cells. The decreased testis coe cient suggests that spermatogenesis may be inhabited, resulting in decreased number of sperm [28]. Interestingly, the index of epididymis markedly increased in the mice treated post exposure (Fig. 2C). It's reported that the increased index means hyperemia or edema of organs while the decreased organ index means atrophy or degenerative changes occurred in organs [29]. The inconsistent results indicate that there may be different response towards exposure to Nano-SiO 2 in testis and epididymis.
As for testis, exposure to Nano-SiO 2 induced vacuolar degeneration of seminiferous tubules, leading to disordered spermatogenesis and decreased quality and quantity of sperm (Fig. 3). It has been veri ed that SiNPs could damage testicular structure, reduce ATP level and affect expressions of regulatory factors of meiosis in the testis of Wistar rats [11]. In addition, SiNPs have been con rmed to pass through the blood-testis barrier (BTB) and the nuclear membranes of spermatocytes in mice through intravenously administration [30]. Based on the previous studies, we speculate that Nano-SiO 2 in mice exposed by intraperitoneal injection could pass through the BTB and be taken up by spermatogenic cells in the luminal compartment of seminiferous tubules directly. Moreover, it's also reported that the in ammation induced by NPs in Leydig cells could reduce testosterone levels, which aggravate the toxicity of NPs on spermatogenic cells through weaken the integrity of the BTB [31]. Therefore, the collapse of seminiferous tubules and the destruction of spermatids in testis could be the direct or indirect effect of NPs.
Epididymis provides a place for sperm maturation which represents a crucial process in male fertility. During this process, sperm acquire both motility and the ability to fertilize. In the present study, lesions of the cauda epididymis were observed after exposure (Fig. 4). The disorganized epithelium and absent of the basement membrane in epididymal tubules implied destruction of blood-epididymis barrier (BEB), which results in stagnation of sperm maturation and post-testicular male infertility (Fig. 5). It is reported that the epididymis in mice is the main target of NPs in the genital area and most of the NPs can reach it through different routes [32]. In male rats, the ratio of the epididymis weight to the body weight increased after gavage with nickel NPs [33]. In another study, the histopathological alterations of epididymis was observed after exposure to Titanium dioxide NPs by oral [34]. Just like in testis, the above abnormality in epididymis could be induced by NPs directly or indirectly. On the organ level, NPs could pass through the barrier and deposit in epididymis, causing irregular cell arrangement and deformed epididymal tubules. On the body level, NPs could change the levels of sex hormones, which is essential for the maintenance of BEB.
As set forth, testosterone is a key factor for the maintenance of male reproductive barriers and fertility. In this study, we found that the concentration of testosterone in serum and expression of genes regulating its biosynthesis in testis were signi cantly decreased in mice 7 days post Nano-SiO 2 (Fig. 6). The change in sex hormone levels after exposure to NPs have been reported in several researches. Intravenous injection of AgNPs in mice could signi cantly increase the concentration of intratesticular testosterone [35], while the serum testosterone level is markedly reduced after treatment with ZO NPs [36]. Moreover, there are even some studies which show that the testosterone and gonadotropin pro les were not altered by CeO 2 NPs or AgNP treatment [37,38]. The disagreement in various studies might be in uenced by different factors, such as particle type, size or the time of exposure. Nevertheless, most of the results showed a decrease in testosterone, which was believed to contribute to severe damage in reproductive organs.
According to previous studies, about 30-80% of male infertility could be attributed to oxidative stressmediated injure [39]. In the present study, the direct measurement of the activity of antioxidant enzyme (SOD) and the level of MDA provided strong evidence for the involvement of oxidative stress caused by Nano-SiO 2 (Fig. 7). Oxidative stress is an important mechanism of the cytotoxic actions of NPs via generating excessive ROS [40]. The imbalance of the oxidative-redox status could cause a wide variety of DNA damage including chromosomal fragmentation, DNA strand breakages and the induction of gene mutation, which nally initiate the process of apoptosis. In our study, we have detected apoptotic cells in testis and epididymis sections post exposure using TUNEL staining (Fig. 9). Indeed, there have been some reports about the NPs mediating apoptosis in somatic cells. A study in human umbilical vein endothelial cells (HUVECs) demonstrated that SiNPs could trigger vascular endothelial cell injure via ROSmediated MAPK/Bcl-2 apoptosis pathway [41]. In another study, JNK-mediated apoptosis pathway was triggered in primary astrocytes by ZnO NPs-induced oxidative stress [42]. Moreover, the results that antioxidative quercetin was able to ameliorate changes in the male reproductive parameters induced by TiO 2 NP suggests that oxidative stress could be a driving force for male fertility [43].Thus, oxidative stress induced apoptosis has been considered as one of the most important causes of male infertility.
It is reported that oxidative stress is linked to in ammation reciprocally in the NPs-induced toxicity [24].
In ammation response is a defensive response of the body to in ammatory factors and local lesions. Our results showed that exposure to Nano-SiO 2 could induce in ammation response in testis and epididymis via stimulating the release of pro-in ammatory mediators, including TNF-α and IL-1β (Fig. 8). It has been reported that in ammation response induced by NPs could cause toxicity and promote cell death through receptor-induced apoptosis [44]. TNF-α, a major production of in ammation response, regulates a diverse range of cellular responses, including cell survival and apoptosis. Indeed, TNFα/TNFR-mediated apoptosis has been veri ed to play a key role in brosis of pneumoconiosis, a class of interstitial lung diseases caused by inhalation of dust containing SiO 2 . It's known that TNF-α could induce apoptosis by activating caspase-8 and -3, but also inhibit apoptosis via overexpression of anti-apoptotic genes such as Bcl-2. In our study, we found that both pro-apoptotic and anti-apoptotic pathway mediated by TNF-α were activated in the testis and epididymis of mice post exposure (Fig. 10). However, the changes we observed in the mice model suggested that the pro-apoptotic pathway may occupy the leading position in the toxicity caused by Nano-SiO 2 . In conclusion, those ndings suggest that the male reproductive system in mice during puberty is sensitive to Nano-SiO 2 exposure. Despite the variation tendency of organ index in testis and epididymis was inconsistent, the abnormality in those organs were both caused by NPs-induced oxidative stress and activation of the apoptosis.

Conclusion
In summary, the present study reveals that intraperitoneally administration of Nano-SiO 2 to pubertal mice could induce oxidative stress and in ammation by disturbing oxidative-redox status and levels of cytokines in testis and epididymis, leading to disordered spermatogenesis and sperm dysmaturity. The oxidative stress and in ammation have been considered as the driving force for augmented male reproductive function caused by NPs, followed by DNA damage and apoptosis. A further study found that TNF-α mediated pro-apoptotic pathway was activated while the anti-apoptotic pathway was inhibited in testis and epididymis post exposure. To the best of our known, this is the rst report about the cytotoxicity of Nano-SiO 2 on male reproductive system during puberty. Those ndings can broaden our understanding of safe applications of Nano-SiO 2 in medicine or other elds, especially for the pubertal male.
Every ve mice were raised in a box and housed at controlled environment (temperature: 23℃; light: 12-h light/dark cycle) with free access to sterilized water and food. After one week acclimation, mice were treated with vehicle (the control group) or Nano-SiO 2 at 40mg/kg dosage per mouse through intraperitoneal injection. At 7 days after injection, the mice were sacri ced and the organs were collected (Fig. S1). All animals experimental protocols were approved by the Committee on the Ethics of Animal Experiments of the North China University of Science and Technology, China (Ethical review number: LAEC-NCST-2020159).

Particle characteristics
Nano-SiO 2 (99.5% trace metals basis, 5-20nm particle size in TEM) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The shape of Nano-SiO 2 was measured by transmission electron microscope (TEM) (JEM-2800, Japan) and the zeta potential in distilled water was detected using a Zetasizer, which re ecting the stability of nanoparticles (NPs) (Zetasizer Mano zs90, UK). Before using, Nano-SiO 2 was vortexed for 1 min and then sonicated for 5 min (120W output; Thermo Scienti c, USA).

Relative reproductive organ weight quanti cation
Body weight was monitored throughout the entire experimental period. After executed, testis and epididymis were collected and weighted. The relative weight of testis (gonadosomatic) and epididymis (epididymal somatic indices) were calculated as the percentage of testicular or epididymal weight in relation to the total body weight.

Daily sperm production
After weighted, the tunica albuginea were removed to release the seminiferous tubules into a homogenizer tube. Then the tissues were homogenized in 1ml of PBS for 2 min and left to stand for 60min at room temperature. Equal volumes of sample were taken to count sperm heads in a haemocytometer chamber. DSP was calculated as: DSP=N/4.48. Where N means the total number of spermatids per sample and 4.48 is a xed coe cient standing for the number of days for a spermatid to develop from stage 14 to stage 16 in mice, which are resistant to homogenization [17].

Histopathological examination
After blood collection, the mice were killed and tissues were rapidly removed and weighted. Unilateral testis and epididymis were xed in Bouin's xing solution, dehydrated in a graded serious of ethanol and xylene solutions, embedded in para n and then sectioned into 5μm sections and mounted onto the glass slides. After stained with hematoxylin and eosin (H&E, BASO), the slides were observed and pictured using optical microscope (Olympus X71, Japan).

Evaluation of sperm parameters
One side of cauda epididymis was dissected from mice after execution, cut into pieces and then put into a 1.5 ml Eppendorf tube containing 200 μl TYH medium at 37℃ for 30 min to allow sperm get out. After incubation, the sperm suspension was divided into two aliquots. One part was diluted with TYH at a ratio of 1:2 and then added to the hemocytometer for counting analysis. The others were washed with PBS and placed on a clean glass slide. After xation with 4% paraformaldehyde, the sperm were stained with Coomassie Brilliant Blue G250 and assessed under high power (×40) of a light microscope to evaluate the sperm morphology. At least 100 spermatozoa were counted [45].

RNA extraction and quantitative Real-time PCR
Testis and epididymis samples were sliced and homogenized in 1 ml Trizol Reagent (Ambition) and then 2ug of total RNA were reverse-transcribed to cDNA using Advantaged RT for PCR Kit (Takara) according to the manufacturers' instructions. cDNAs were subsequently subjected to real-time PCR analysis using speci c primers synthesized by Sangon Biotech (Beijing, China) listed in Table S2 with a 2X M5 HiPer Realtime PCR Super mix (Mei5bio). A melting curve analysis was performed to exclude nonspeci c product ampli cation. The expression levels of the target genes were normalized to Actb content, and the relative fold change were calculated based on the 2 -ΔΔCt comparative method [46].

Oxidative stress measurement
Total proteins in testis and epididymis tissues were extracted in PBS containing protease inhibitor cocktail (P8340, Sigma) with an ultrasonic cell crusher on ice. The supernatants were collected and then added to the reagents for measuring the levels of MDA and SOD according to the instructions of the oxidation-antioxidation assay kits (A003-1, A001-3, Jiancheng).

TUNEL assay
Apoptotic cells on testis or epididymal para n sections were detected using One Step TUNEL Assay Kit (Beyotime) according to the manufacturer's protocol. Images were taken with a Nikon uorescent microscope.
Testosterone levels Blood was collected through avulsion of the eyeball, incubation at room temperature for 30 min and then centrifuged at 5000 rpm for 30 min. The supernatant was collected as serum and stored at -80℃ until analysis. The levels of testosterone in serum were diluted with phosphate buffered saline (PBS) at the ratio of 1:4 and assayed in triplicate using speci c enzyme-linked immunosorbent assay (ELISA) commercial kits according to the manufacturers' protocol (Cayman). The concentration of each sample was determined using the equation obtained from the standard curve plot. All samples were examined twice when fell out the standard curve. The variation coe cients of inter-assay were 2.8-7.7% [17].

Statistical analysis
All results are presented as means ± standard error (SE) (n≥3) and examined for their statistical signi cance of differences by Student's t-test. All comparisons were considered signi cantly different when p-value < 0.05.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval
All animal experiments were reviewed and approved by the Laboratory Animal Ethics Committee of North China University of Science and Technology.

Consent for publication
Not applicable.       Malondialdehyde (MDA) assay kit in testicular and epididymal tissue is signi cantly increased after exposure. Data was presented as mean ± SE of at least three mice per group. *p < 0.05 compared with control.

Figure 8
In ammation response is induced by Nano-SiO2 in the testis and epididymis. Real-time PCR assessment of mRNA levels for in ammatory cytokines ( TNF-α, IL-1β) shows a remarkable increase in the expression Page 24/25 of those genes. The housekeeping gene Actb is used for expression normalization. Data is presented as mean ± SE of at least three mice per group. *p < 0.05 versus control.

Figure 9
Nano-SiO2 induces DNA-damage in testis and epididymis which leading to cell apoptosis. TUNEL staining is used to detect the apoptotic cells in the para n sections of testis and epididymis, the results show that there is a signi cant increase of apoptotic cells in mice treated with Nano-SiO2. Images are captured from two independent experiments and the pictures showed in the gure are representative. TNF-α mediated pro-apoptotic pathway is activated in the testis and epididymis post exposure. Mice exposed to Nano-SiO2 exhibit an increase in protein levels of TNF-α mediated pro-apoptotic genes (TNF-R , NF-κB, cleaved-Caspase8, cleaved-Caspase3) and anti-poptotic genes (Bcl-2) in testicular (A, B) and epididymal tissues (C, D) as determined with Western blot. *p < 0.05, n = 3 each group.

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