BECLIN-1-Mediated Autophagy Suppresses Silica Nanoparticle-Induced Testicular Toxicity via the Inhibition of Caspase 8-Mediated Cell Apoptosis in Leydig Cells

Accumulation of silica nanoparticles (SNPs) in the testes leads to male reproductive toxicity. However, little is known about the effect and mechanistic insights of SNP-induced autophagy on apoptosis in Leydig cells. In this study, we aimed to verify the role of SNP-induced autophagy in apoptosis and explore the possible underlying mechanism in mouse primary Leydig cells (PLCs). H&E staining showed that SNPs changed the histological structures of the testes, including a reduction in the Leydig cell populations in vivo. CCK-8 assay showed that SNPs decreased cell viability, and flow cytometry showed that SNPs increased cell apoptosis, both in a dose-dependent manner in vitro. Additionally, Western blotting further found that SNPs activated autophagy by an increase in BECLIN-1, ATG16L, and LC3-II levels and promoted the intrinsic pathway of apoptosis by an increase in the BAX/BCL-2 ratio, cleaved the caspase 8 and caspase 3 levels. Furthermore, autophagy decreased SNP-induced apoptosis via regulation of the caspase 8 level combined with rapamycin, 3-methyladenine, and chloroquine. BECLIN-1 depletion increased the caspase 8 level, leading to an increase in SNP-induced cell apoptosis. Collectively, this evidence demonstrates that SNPs activated BECLIN-1-mediated autophagy, which prevented SNP-induced testicular toxicity via the inhibition of caspase 8-mediated cell apoptosis in Leydig cells.


Introduction
Silica nanoparticles (SNPs), one of the readily available nanomaterials, have been widely used in agriculture, cosmetics, food additives, and biomedical applications [1]. Owing to their wide applicability, especially in consumer products and as an additive in biomedical formulations, the risk of human and animal exposure needs to be investigated [2,3]. Depending on the physicochemical properties and duration and route of exposure, SNPs can potentially cause toxicity in a variety of organs, such as liver, kidneys, brain, lungs, ovaries, and testes [4][5][6]. For example, SNPs can cross the blood-testis barrier (BTB) and distribute in testicular tissue, resulting in testicular toxicity [6][7][8].
nium hydroxide (26 mmol) were mixed in a flask with a stirring rate of 400 rpm for 10 min. Next, TEOS (16 mmol) was added dropwise, and the reaction was left under stirring for 24 h at room temperature. The synthesized SNPs were pelleted and precipitated by centrifugation using an Avanti J-15R centrifuge (Beckman Coulter Inc., Indianapolis, IN, USA) with a stirring rate of 15,000 rpm for 20 min, washed with distilled water and 95% ethanol three times, and stored in absolute ethanol at a concentration of 4 mg/mL. Before use, the stock solution was recentrifuged at 18,000 rpm for 30 min and redispersed in sterilized water. The physicochemical characteristics of SNPs were analyzed by transmission electron microscopy (TEM; Tecnai 12; Royal Philips, Amsterdam, The Netherlands) and a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK).

Animals and Treatment with SNPs
Male C57BL/6 mice (8-week-old, 20.0 ± 2.0 g) were obtained from the Comparative Medicine Center of Yangzhou University and maintained under standard conditions at the temperature range of 23-25 • C, 55-60% humidity. and a 12 h light/dark cycle.
The mice were provided with about 5 g of food and unlimited purified water and were randomly divided into a control group and an SNP treatment group. On the first day, the control group was administered with 0.2 mL of saline via tail vein injection (n = 6). The SNP treatment group was administered with 25 mg/kg of SNPs via tail vein injection (n = 6) as described previously [4]. After injection, the mice were weighed and sacrificed after 7 days. The testes were removed from the abdominal cavities and immersed in a modified Davidson's fluid tissue fixative (including 95% alcohol, formaldehyde, glacial acetic acid, and ultrapure water in a ratio of 3:2:1:2) for hematoxylin-eosin (H&E) staining.

H&E Staining
The fixed testis samples were dehydrated by graded ethanol (50%, 75%, 80%, 95%, and absolute ethanol for 1 h each), placed in xylene until transparent, and then embedded in paraffin; 5 µm thick sections were cut continuously for subsequent staining. The sections were deparaffinized in xylene and hydrated with graded ethanol (absolute ethanol I and II for 5 min, respectively; 95% alcohol I and II for 3 min, respectively; 80% and 70% for 3 min). Next, the tissue sections were stained with hematoxylin for 3 min, washed with water, and stained in eosin for 12 s. After cleaning with xylene and drying, the sections were mounted with neutral balsam. The morphological structure was observed by digital microscopy (BA400, Motic, Amoy, Xiamen, China).

PLC Culture In Vitro and Treatment with SNPs
The testes were removed from 8-week-old male mice and collected in DMEM/F12. After decapsulation, they were added to DMEM/F12 containing 1 mg/mL of collagenase I to separate PLCs from other testicular cells and incubated in a shaking water bath at 37 • C for 10 min. After removing the floc of the testes, the same amount of DMEM/F12 with 10% FBS was added to stop digestion. After filtration, the PLCs were distributed in a culture medium inside 60 mm tissue culture dishes containing DMEM/F12 with 10% FBS and 100 mg/L of penicillin/streptomycin at 37 • C and 5% CO 2 . After 6 h, the medium was removed and replaced with fresh DMEM/F12. When cell confluency reached 70-80%, the PLCs were treated with SNPs.

Determination of SNP Uptake
The SNP-treated PLCs were carefully washed in 0.01 M PBS (pH = 7.4), fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in PBS for 24 h. The PLCs were washed with PBS three times, embedded in agarose gel (2%), postfixed in osmium tetroxide (4%), and washed again with PBS. Next, the samples were dehydrated with gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) and embedded in epoxy resin. The sections were cut using an ultramicrotome and stained with uranyl acetate-lead citrate (3%). Finally, the ultrastructure of the PLCs was observed by TEM.

Cell Viability Assay
Cell viability of the PLCs was assessed after SNP treatment using Cell Counting Kit 8 (CCK-8, New Cell and Molecular Biotech Co. Ltd., Suzhou, China); 3 × 10 4 cells/200 µL medium/well were seeded in 96-well plates for 24 h and then treated with various doses of SNPs (control, 100, 200, 400, 600, 800, 1000, and 1200 µg/mL) for 24 h; 10 µL of CCK-8 were added into each well and incubated for 2 h at 37 • C. Finally, the absorbance readings were taken at 450 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Model 680, Bio-Rad, and Hercules, CA, USA).

Cell Apoptosis Assay
Cell apoptosis in the PLCs was determined after SNP treatment by flow cytometry (EPICddmics Altra, Brea, CA, USA); 2 × 10 6 PLCs/well were seeded into a 6-well culture plate for 24 h and then treated with various concentrations of SNPs (control, 200, 400, and 800 µg/mL) for 24 h. The cells were washed twice with PBS, trypsinized, centrifuged, and harvested. After resuspension in 500 µL of a binding buffer, 5 µL of Annexin V-FITC or V-PE and propidium iodide (PI) (Nanjing KeyGen Biotech, Nanjing, China) were added into the cells. The cell apoptotic rate was determined within 1 h by flow cytometry.

Monodansylcadaverine (MDC) Staining Detection
MDC staining was performed to monitor the activation of autophagy in the PLCs after SNP treatment as described previously [26]. Briefly, the PLCs were cultured on sterile cover slips and then treated with various doses of SNPs for 12 h. Next, the PLCs were incubated with MDC solution in the dark. Finally, the PLCs were examined by laser scanning confocal microscopy (TCS SP8 STED; Wetzlar, Hessen, Germany).

Statistical Analysis
All the statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software (version 18.0; SPSS, Chicago, IL, USA). An independent-samples t-test was used to compare two groups. One-way ANOVA was used to analyze more than two groups followed by the least significant difference (LSD) test to compare various groups. All the values are presented as the means ± SEM from independent experiments performed in triplicate, and the difference was statistically at p < 0.05.

Characterization of SNPs
The morphology of the Stöber SNPs was determined by TEM. Low-magnification TEM images showed the SNP particles to be of spherical shape with low polydispersity ( Figure 1A). The average diameter of SNPs was 109.0 ± 14.5 nm which was determined by randomly counting and measuring more than 500 particles ( Figure 1B). The hydrodynamic diameter of SNPs and the zeta potential of SNPs can be obtained from our previous study [26].
cial Sciences (SPSS) software (version 18.0; SPSS, Chicago, IL, USA). An independent-samples t-test was used to compare two groups. One-way ANOVA was used to analyze more than two groups followed by the least significant difference (LSD) test to compare various groups. All the values are presented as the means ± SEM from independent experiments performed in triplicate, and the difference was statistically at p < 0.05.

Characterization of SNPs
The morphology of the Stöber SNPs was determined by TEM. Low-magnification TEM images showed the SNP particles to be of spherical shape with low polydispersity ( Figure 1A). The average diameter of SNPs was 109.0 ± 14.5 nm which was determined by randomly counting and measuring more than 500 particles ( Figure 1B). The hydrodynamic diameter of SNPs and the zeta potential of SNPs can be obtained from our previous study [26].
A B

SNPs Change the Histological Structures of Testes In Vivo
To observe the toxic effects of SNPs on testes in vivo, the histological structures of the testes were observed by H&E staining. The results showed that the testes from the control group had normal testicular architecture and germinal cell arrangement ( Figure  2A-D). The Leydig cells and interstitial tissue had the same size and their count was normal ( Figure 2D). All the spermatogenic cells and Sertoli cells were normal ( Figure  2D). However, the testes from the SNP group showed marked atrophy and deformation of the seminiferous tubules characterized by a reduction in the Leydig cell populations and disorganization of spermatogenic cell layers ( Figure 2E-H).

SNPs Change the Histological Structures of Testes In Vivo
To observe the toxic effects of SNPs on testes in vivo, the histological structures of the testes were observed by H&E staining. The results showed that the testes from the control group had normal testicular architecture and germinal cell arrangement (Figure 2A-D). The Leydig cells and interstitial tissue had the same size and their count was normal ( Figure 2D). All the spermatogenic cells and Sertoli cells were normal ( Figure 2D). However, the testes from the SNP group showed marked atrophy and deformation of the seminiferous tubules characterized by a reduction in the Leydig cell populations and disorganization of spermatogenic cell layers ( Figure 2E-H).

SNP Cellular Uptake in Primary Leydig Cells (PLCs)
To determine whether SNPs can be taken up by PLCs, the localization of SNPs within the PLCs was monitored by TEM. The results showed that there were no particles in the control group ( Figure 3A

SNP Cellular Uptake in Primary Leydig Cells (PLCs)
To determine whether SNPs can be taken up by PLCs, the localization of SNPs within the PLCs was monitored by TEM. The results showed that there were no particles in the control group ( Figure 3A

SNP Cellular Uptake in Primary Leydig Cells (PLCs)
To determine whether SNPs can be taken up by PLCs, the localization of SN within the PLCs was monitored by TEM. The results showed that there were no partic in the control group ( Figure 3A

SNPs Decreased Cell Viability and Increased Cell Apoptosis in the PLCs
To determine the effect of SNPs on cell viability and apoptosis, CCK-8 and flow cytometry were employed to analyze the samples after different doses of SNP treatment for 24 h. The CCK-8 assay showed that SNPs (above 200 µg/mL) decreased cell viability in a dose-dependent manner ( Figure 4A). The flow cytometry assay showed that SNPs increased cell apoptosis in a dose-dependent manner ( Figure 4B,C). The apoptotic rate was significantly increased in the groups after 200, 400, and 800 µg/mL SNP treatments (10.70 ± 1.17%, 15.83 ± 1.63%, and 24.42 ± 2.58%, respectively) compared to the control group (6.01 ± 0.63%) ( Figure 4B,C).

SNPs Decreased Cell Viability and Increased Cell Apoptosis in the PLCs
To determine the effect of SNPs on cell viability and apoptosis, CCK-8 and flow cytometry were employed to analyze the samples after different doses of SNP treatment for 24 h. The CCK-8 assay showed that SNPs (above 200 µg/mL) decreased cell viability in a dose-dependent manner ( Figure 4A). The flow cytometry assay showed that SNPs increased cell apoptosis in a dose-dependent manner ( Figure 4B,C). The apoptotic rate was significantly increased in the groups after 200, 400, and 800 µg/mL SNP treatments (10.70 ± 1.17%, 15.83 ± 1.63%, and 24.42 ± 2.58%, respectively) compared to the control group (6.01 ± 0.63%) ( Figure 4B,C).

SNPs Activated Autophagy and the Apoptotic Pathway in the PLCs
To ensure whether SNPs activate autophagy and the intrinsic pathway of apoptosis, MDC staining and Western blotting were employed to analyze the samples after different doses of SNP treatment for 12 h. MDC staining showed that SNPs enhanced the accumulation of autophagic vacuoles in the PLCs in a dose-dependent manner ( Figure  5A). The fluorescent intensity was significantly increased in the groups after 200, 400,

SNPs Activated Autophagy and the Apoptotic Pathway in the PLCs
To ensure whether SNPs activate autophagy and the intrinsic pathway of apoptosis, MDC staining and Western blotting were employed to analyze the samples after different doses of SNP treatment for 12 h. MDC staining showed that SNPs enhanced the accumulation of autophagic vacuoles in the PLCs in a dose-dependent manner ( Figure 5A). The fluorescent intensity was significantly increased in the groups after 200, 400, and 800 µg/mL SNP treatments compared to the control group ( Figure 5A). Furthermore, Western blotting showed that the levels of ATG16L, BECLIN-1, LC3-II, the BAX/BCL-2 ratio, cleaved caspase 8, and cleaved caspase 3 were significantly increased in a certain dose-dependent manner ( Figure 5B-E). The P62 level was significantly increased in the group after 800 µg/mL SNP treatment and had no significant difference between the groups after 200 and 400 µg/mL SNP treatments compared to the control group ( Figure 5B,C). The ATG5 and ATG4B levels had no significant difference between the SNP and control groups ( Figure 5B,C). The BAX/BCL-2 ratio was significantly increased in a dose-dependent manner; cleaved caspase 8 and cleaved caspase 3 were also significantly increased in the groups after 400 and 800 µg/mL SNP treatments and had no significant difference between the groups after 200 µg/mL SNP treatments compared to the control group ( Figure 5D,E) in a certain dose-dependent manner ( Figure 5B-E). The P62 level was significantly increased in the group after 800 µg/mL SNP treatment and had no significant difference between the groups after 200 and 400 µg/mL SNP treatments compared to the control group ( Figure 5B,C). The ATG5 and ATG4B levels had no significant difference between the SNP and control groups ( Figure 5B,C). The BAX/BCL-2 ratio was significantly increased in a dose-dependent manner; cleaved caspase 8 and cleaved caspase 3 were also significantly increased in the groups after 400 and 800 µg/mL SNP treatments and had no significant difference between the groups after 200 µg/mL SNP treatments compared to the control group ( Figure 5D

Autophagy Inhibited SNP-Induced Apoptosis in the PLCs
To investigate the effect of autophagy on SNP-induced apoptosis, flow cytometry and Western blotting were employed to analyze the samples after SNP treatment combined with PBS (control), early autophagy inhibitor 3-MA, late inhibitor CQ, and activator Rap. The flow cytometry assay showed that the activation of autophagy decreased SNP-induced apoptosis, whereas the inhibition of autophagy increased SNP-induced apoptosis in the PLCs ( Figure 6A,B). The apoptotic rate was increased in the 3-MA + SNP (19.38 ± 2.08%) and the CQ + SNP (22.04 ± 2.46%) groups, whereas it was decreased in the RAP + SNP group (10.25 ± 1.24%) compared to the PBS + SNP group (14.51 ± 1.65%) ( Figure 6A,B). Western blotting showed that pretreatment with 3-MA decreased the BECLIN-1 and LC3-II levels, whereas it increased the BAX/BCL-2 ratio and the cleaved caspase 8 and cleaved caspase 3 levels compared to the PBS + SNP group ( Figure  6C-Fc). Pretreatment with CQ decreased the BECLIN-1 level, whereas it increased P62, LC3-II, the BAX/BCL-2 ratio, and the cleaved caspase 8 and cleaved caspase 3 levels compared to the PBS + SNP group ( Figure 6C-Fc). Pretreatment with RAP increased the BECLIN-1 and LC3-II levels, whereas it decreased P62, the BAX/BCL-2 ratio, and the cleaved caspase 8 and cleaved caspase 3 levels compared to the PBS + SNP group ( Figure  6C-Fc).

Autophagy Inhibited SNP-Induced Apoptosis in the PLCs
To investigate the effect of autophagy on SNP-induced apoptosis, flow cytometry and Western blotting were employed to analyze the samples after SNP treatment combined with PBS (control), early autophagy inhibitor 3-MA, late inhibitor CQ, and activator Rap. The flow cytometry assay showed that the activation of autophagy decreased SNP-induced apoptosis, whereas the inhibition of autophagy increased SNP-induced apoptosis in the PLCs ( Figure 6A,B). The apoptotic rate was increased in the 3-MA + SNP (19.38 ± 2.08%) and the CQ + SNP (22.04 ± 2.46%) groups, whereas it was decreased in the RAP + SNP group (10.25 ± 1.24%) compared to the PBS + SNP group (14.51 ± 1.65%) ( Figure 6A,B). Western blotting showed that pretreatment with 3-MA decreased the BECLIN-1 and LC3-II levels, whereas it increased the BAX/BCL-2 ratio and the cleaved caspase 8 and cleaved caspase 3 levels compared to the PBS + SNP group ( Figure 6C-Fc). Pretreatment with CQ decreased the BECLIN-1 level, whereas it increased P62, LC3-II, the BAX/BCL-2 ratio, and the cleaved caspase 8 and cleaved caspase 3 levels compared to the PBS + SNP group ( Figure 6C-Fc). Pretreatment with RAP increased the BECLIN-1 and LC3-II levels, whereas it decreased P62, the BAX/BCL-2 ratio, and the cleaved caspase 8 and cleaved caspase 3 levels compared to the PBS + SNP group ( Figure 6C-Fc).

Discussion
Numerous studies demonstrate that SNPs can cause toxicity in the male reproductive system [28][29][30][31]. SNPs can accumulate in testes, damage seminiferous tubules and Leydig cells, and inhibit spermatogenesis, in turn resulting in a decrease in the quality and quantity of sperms via intravenous [8], intramuscular [32], or inhalation [6] exposure routes. Leydig cells play an essential role in promoting spermatogenesis as they help in testosterone synthesis and secretion. However, the mechanisms of Leydig cell damage have not been studied. In this study, we demonstrated that SNPs caused cell apoptosis in mouse Leydig cells and activated BECLIN-1-mediated autophagy. Furthermore, BECLIN-1-mediated autophagy prevented SNP-induced cytotoxicity via the inhibition of the mitochondriamediated apoptotic pathway and caspase-8-mediated death signaling, which provided evidence that autophagy and apoptosis in Leydig cells result in SNP-induced toxicity.
In this study, we treated cells with RAP to activate autophagy and with 3-MA and CQ to inhibit autophagy prior to treatment with SNPs. We found that SNP-induced BECLIN-1 and LC3-II levels were further significantly increased and the P62 level was decreased, whereas BECLIN-1 and LC3-II were decreased by 3-MA, and P62 and LC3-II were significantly increased by CQ via the inhibition of autophagic flux ( Figure 6). The results are in good agreement with previous studies, where Xi et al. found that RAP increased the BECLIN-1 and LC3-II levels, whereas 3-MA inhibited the BECLIN-1 and LC3-II levels combined with mesoporous spherical SNPs (150 nm) in RAW 264.7 macrophages [24]. Ha et al. found that 3-MA inhibited the LC3-II level, whereas CQ increased the P62 and LC3-II levels combined with mesoporous spherical SNPs (50 nm) in MC3T3-E1 cells [43]. The evidence above shows that SNPs can enhance BECLIN-1-mediated autophagy at the dosages investigated.
To determine the effect of BECLIN-1-mediated SNP-activated autophagy on cell apoptosis in PLCs, SNP-induced cell apoptosis was inhibited by RAP, whereas it was enhanced by 3-MA and CQ ( Figure 6). Consistent with a previous study, SNPs (236 nm) activated autophagy while protecting RAW 264.7 macrophages from cell death, and 3-MA combined with SNPs not only increased cell death, but also resulted in cell membrane integrity loss in RAW 264.7 macrophages [23]. Furthermore, we determined the levels of apoptosis-related proteins. We found that SNP-induced cleaved caspase 8, the BAX/BCL-2 ratio, and cleaved caspase 3 levels were significantly decreased by RAP, whereas they were increased by 3-MA and CQ ( Figure 6). We knocked down BECLIN-1 by RNA interference to block autophagy. Compared with SNPs alone, the inhibition of autophagy by knockdown of BECLIN-1 significantly decreased the BECLIN-1 and LC3-II levels, whereas it increased the cleaved caspase 8, the BAX/BCL-2 ratio, and cleaved caspase 3 levels (Figure 7). Some recent studies have shown that SNPs induced autophagy and apoptosis [45], but only a few of them elucidated the relationship between autophagy and apoptosis. Our findings indicate that the activation of BECLIN-1-mediated autophagy inhibits SNP-induced cell apoptosis in Leydig cells.
BECLIN-1, a novel BCL-2 homology (BH)-3 domain-only protein [17], can interact with antiapoptotic protein BCL-2 via its BH3 domain [16]. However, unlike other known BH3-only proteins, BECLIN-1 does not exert a proapoptotic function [46]. In contrast, BECLIN-1 plays an antiapoptotic role in several conditions, such as nutrient deprivation [47], hypoxia [48], and nanoparticle exposure [49]. BCL-2 interacts with the BH3 domain of BECLIN-1, resulting in inhibited autophagy activation, where both autophagy blockage and autophagy gene depletion can cause cell apoptosis under stress stimulation, whereas downregulation of BCL-2 leads to the activation of autophagy [18]. In this study, we found that at the concentrations studied, Stöber SNPs increase the BECLIN-1 level and decrease the BCL-2 level ( Figure 5), thus promoting BECLIN-1-mediated critical complex formation in autophagy initiation. Furthermore, we found that BECLIN-1 depletion inhibited autophagy activation via the decrease of the LC3-II level, indicating that BECLIN-1 played an important role in SNP-induced autophagy initiation. It has been reported that SNPs can induce spermatocyte cell apoptosis via caspase 8-mediated cell death pathway [50,51]. In this study, we found that SNPs could increase the cleaved caspase 8 level (Figure 5), indicating caspase 8-mediated cell death cooperated in SNP-induced cell toxicity in the PLCs. The activation of BECLIN-1-mediated autophagy by RAP decreased the cleaved caspase 8 level, while the inhibition of BECLIN-1-mediated autophagy by 3-MA and CQ increased the cleaved caspase 8 level, indicating that cleaved caspase 8 could be regulated by BECLIN-1-mediated autophagy ( Figure 6). These findings are consistent with a previous report where the authors demonstrated that cleaved caspase 8 can be degraded by autophagy under conditions associated with cytoprotective autophagy [19]. Liu et al. demonstrated that BECLIN-1-mediated autophagy protects against cadmium-induced activation of apoptosis via the interaction of BECLIN-1 with cleaved caspase 8 to impair its proapoptotic activation [20]. Caspase 8 not only is involved in death receptor-mediated apoptosis, but also cooperates in mitochondria-mediated apoptosis. Caspase 8 and Bid can interact with mature cardiolipin of the mitochondria, resulting in the initiation of early apoptotic signaling [52]. Our study showed that BECLIN-1 depletion increased the cleaved caspase 8 level (Figure 7), further indicating that BECLIN-1-mediated autophagy protects from SNP-induced cell apoptosis via the inhibition of caspase 8 in PLCs.
Autophagy is a double-edged sword for cell survival and death. SNPs can be taken up by cells via endocytosis and are sequestrated and accumulated by autophagosomes or lysosomes to degrade them. The intensity of the autophagic response is influenced by size, charge, dose, and treatment time of SNPs [41]. Positively charged and larger SNPs (≥100 nm) during higher treatment doses (≥500 µg/mL) and longer time (24 h) can induce autophagy, resulting in autophagy-mediated cell death [41]. Hence, SNPs are able to induce an increase in autophagosome formation and flux and autophagic dysfunction. The LC3-II protein is increased in both categories; however, P62 is only increased in the case of autophagic dysfunction, which is no longer degraded via autophagy [53]. For example, Wang et al. showed that high doses of 60 nm and 16 nm of nonporous spherical SNPs inhibited autophagosome degradation, leading to autophagy dysfunction via detecting the P62 level increase in HepG2 cells and BEAS-2B cells, respectively [54,55]. In this study, we found that 800 µg/mL of SNPs also increased the P62 level, indicating that high doses of SNPs inhibited autophagosome degradation, resulting in autophagy dysfunction in the PLCs ( Figure 5), which would be determined in a further study. In summary, SNPs either induced or disrupted autophagy in connection with cell type, particle dose, and treatment time. These doses may or may not be relevant to human exposure depending on the extent and route of exposure.
The findings from this study demonstrate that SNPs cause cell apoptosis in Leydig cells, which provides a possible mechanism of SNP-caused testicular toxicity. Furthermore, BECLIN-1-mediated autophagy inhibits SNP-induced cytotoxicity in Leydig cells via the inhibition of the mitochondria-mediated apoptotic pathway and even the death receptor-mediated death signaling, which provides a strategy to relieve or abate SNPcaused testicular toxicity via supplementary autophagy activators. A diagram of the mechanisms involved in SNP-induced cytotoxicity in PLCs summarizes these findings as shown in Figure 8. example, Wang et al. showed that high doses of 60 nm and 16 nm of nonporous spherical SNPs inhibited autophagosome degradation, leading to autophagy dysfunction via detecting the P62 level increase in HepG2 cells and BEAS-2B cells, respectively [54,55]. In this study, we found that 800 µg/mL of SNPs also increased the P62 level, indicating that high doses of SNPs inhibited autophagosome degradation, resulting in autophagy dysfunction in the PLCs (Figure 5), which would be determined in a further study. In summary, SNPs either induced or disrupted autophagy in connection with cell type, particle dose, and treatment time. These doses may or may not be relevant to human exposure depending on the extent and route of exposure.
The findings from this study demonstrate that SNPs cause cell apoptosis in Leydig cells, which provides a possible mechanism of SNP-caused testicular toxicity. Furthermore, BECLIN-1-mediated autophagy inhibits SNP-induced cytotoxicity in Leydig cells via the inhibition of the mitochondria-mediated apoptotic pathway and even the death receptor-mediated death signaling, which provides a strategy to relieve or abate SNP-caused testicular toxicity via supplementary autophagy activators. A diagram of the mechanisms involved in SNP-induced cytotoxicity in PLCs summarizes these findings as shown in Figure 8.

Conclusions
In summary, SNPs are able to activate autophagy via the enhancement of autophagosome formation, extension, and maturation and induce cell apoptosis via the intrinsic

Conclusions
In summary, SNPs are able to activate autophagy via the enhancement of autophagosome formation, extension, and maturation and induce cell apoptosis via the intrinsic apoptotic pathway in Leydig cells. Meanwhile, the activation of autophagy protects Leydig cells from SNP-induced apoptosis via the inhibition of the mitochondria-mediated apoptotic pathway and even the death receptor-mediated death signaling.