Exposure to TiO2 Nanoparticles Induces Immunological Dysfunction in

Article pubs.acs.org/JAFC

Exposure to TiO2 Nanoparticles Induces Immunological Dysfunction in Mouse Testitis Fashui Hong,*,†,‡,§,∥ Yajing Wang,†,‡,§,∥ Yingjun Zhou,†,‡,§,∥ Qi Zhang,†,‡,§ Yushuang Ge,†,‡,§ Ming Chen,†,‡,§ Jie Hong,# and Ling Wang⊥ †

Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection, Huaiyin Normal University, Huaian 223300, China ‡ Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake, Huaiyin Normal University, Huaian 223300, China § School of Life Sciences, Huaiyin Normal University, Huaian 223300, China # Medical College of Soochow University, Suzhou 215123, China ⊥ Library of Soochow University, Suzhou 215123, China ABSTRACT: Although TiO2 nanoparticles (NPs) as endocrine disruptors have been demonstrated to be able to cross the blood−testis barriers and induce reproductive toxicity in male animals, whether the reproductive toxicity of male animals due to exposure to endocrine disruptor TiO2 NPs is related to immunological dysfunction in the testis remains not well understood. This study determined whether the reproductive toxicity and immunological dysfunction induced by exposure to TiO2 NPs is associated with activation or inhibition of TAM/TLR-mediated signal pathway in mouse testis. The results showed that male mice exhibited significant reduction of fertility, infiltration of inflammatory cells, rarefaction, apoptosis, and/or necrosis of spermatogenic cells and Sertoli cells due to TiO2 NPs. Furthermore, these were associated with decreased expression of Tyro3 (−18.16 to −66.6%), Axl (−14.7 to −57.99%), Mer (−7.98 to −72.62%), and IκB (−11.25 to −63.16%), suppression of cytokine signaling (SOCS) 1 (−21.99 to −73.8%) and SOCS3 (−8.11 to −34.86%), and increased expression of Toll-like receptor (TLR)-3 (21.4−156.03%), TLR-4 (37.0−109.87%), nuclear factor-κB (14.75−69.34%), interleukin (IL)-lβ (46.15−123.08%), IL-6 (2.54−81.98%), tumor necrosis factor-α (6.95−88.39%), interferon (IFN)-α (2.54−37.25%), and IFN-β (10.19−80.56%), which are involved in the immune environment in the testis. The findings showed that reproductive toxicity of male mice induced by exposure to endocrine disruptor TiO2 NPs may be associated with biomarkers of impairment of immune environment or dysfunction of TAM/TLR3-mediated signal pathway in mouse testitis. Therefore, the potential risks to reproductive health should be attended, especially in those who are occupationally exposed to TiO2 NPs. KEYWORDS: titanium dioxide nanoparticles, reproductive toxicity, testitis, immune dysfunction, TAM/TLRs signal pathway

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INTRODUCTION Nano-sized materials (NMs) have been widely used in various areas. Among the varieties of engineered NMs being used today, TiO2 nanoparticles (TiO2 NPs) occupy a prominent position. For example, TiO2 NPs have been widely used in various industries including painting, printing, and medicine and environmental protection, food, and cosmetics, etc.1−5 The use of TiO2 NPs had increased in today’s largest production and, meanwhile, have caused a growing concern for the risks to human health. Indeed, although bulk TiO2 has been classified as a biologically inert material for humans and animals,6 evidence has been repeatedly reported on TiO2 NPs toxicity in animals in vivo, with inflammation of liver, kidney, spleen, lung, and brain7−12 and carcinogenicity.13 Because of their potential toxicity, the NIOSH issued a recommended exposure limit (REL) of 0.3 mg/m3 for ultrafine TiO2 particles and concluded that ultrafine TiO2 (including engineered nanoscale) is a potential occupational carcinogen.14 As is known, reproductive organs as endocrine systems are very sensitive to stress such as heavy metals, xenobiotic compounds, microwaves, and NPs. The extensive applications of NPs have inevitably led to harmful biological responses in the reproductive system or endocrine system.15,16 Numerous © 2015 American Chemical Society

studies have demonstrated that NPs are able to cross the blood−testes and blood−brain barriers and accumulate in the testis or brain due to their nanoscale size,17,18 and these NPs induced cytotoxicity and gene expression changes, leading to impairment of the male mouse reproductive system.19,20 It has been reported that prenatal exposure to TiO2 NPs (25−70 nm, 20−25 m2/gin surface area, anatase) caused male offspring with altered spermatogenesis and histopathological changes and was translocated to spermatids, Sertoli cells, and Leydig cells, resulting in disorganization in seminiferous tubules and reductions in daily sperm production, sperm motility, and Sertoli cell number in the male mouse offspring testes.21 Prenatal exposure to rutile TiO2 NPs (primary diameter = 35 nm, agglomerates averaging 193.3 nm in diameter) also led to deposition of TiO2 NPs and Sertoli cell and sperm damages in the male mouse offspring testes.22 Previous studies observed that male mouse exposure to TiO2 NPs (without indicating characterization) led to significant reductions in sperm numbers Received: Revised: Accepted: Published: 346

November 3, 2015 December 18, 2015 December 19, 2015 December 31, 2015 DOI: 10.1021/acs.jafc.5b05262 J. Agric. Food Chem. 2016, 64, 346−355

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Journal of Agricultural and Food Chemistry Table 1. Real-Time PCR Primer Pairs: PCR Primers Used in the Gene Expression Analysis gene name

description

refer-GAPDH

mGAPDH-F mGAPDH-R

5′-TGTGTCCGTCGTGGATCTGA-3′ 5′-TTGCTGTTGAAGTCGCAGGAG-3′

150

mactin-F mactin -R

5′-GAGACCTTCAACACCCCAGC-3′ 5′-ATGTCACGCACGATTTCCC-3′

263

mTyro3-F mTyro3-R

5′-GGTCGGATGTTGGGCAAAG-3′ 5′-CGGAGGAACTCTTCTATGTCGC-3′

140

mAxl-F mAxl-R

5′-GACTCCATCCTCAAGGTCGC-3′ 5′-CCTGAAAACAGACGCCAATG-3′

mMer-F mMer-R

5′-TCTGCGTGGCAGACTTTGG-3′ 5′-CACTTTTGCTTGTGTAGACTCGG-3′

135

mNF-κB-F mNF-κBR

5′-GTGGAGGCATGTTCGGTAGTG-3′ 5′-TCTTGGCACAATCTTTAGGGC-3′

195

mIκB-F mIκB-R

5′-GGTGCAGGAGTGTTGGTGG-3′ 5′-TGGCTGGTGTCTGGGGTAC-3′

173

mTLR-3-F mTLR-3-R

5′-ATGATGTCGGCAACGGTTC-3′ 5′-CACTTTGCTTAGTAAATGCTCGC-3′

153

mTLR-4-F mTLR-4-R

5′-CATCAGTGTGTCAGTGGTCAGTG-3′ 5′-CTCATTTCTCACCCAGTCCTCA-3′

175

mIL-1β-F mIL-1β-R

5′-GCTTCAGGCAGGCAGTATCA-3′ 5′-TGCAGTTGTCTAATGGGAACG-3′

196

mIL-6-F mIL-6-R

5′-GGGACTGATGCTGGTGACAAC-3′ 5′-CAACTCTTTTCTCATTTCCACGA-3′

163

mTNF-α-F mTNF-α-R

5′-CCCTCCAGAAAAGACACCATG-3′ 5′-CACCCCGAAGTTCAGTAGACAG-3′

183

mIFN-β-F mIFN-β-R

5′-TGCGTTCCTGCTGTGCTTC-3′ 5′-CGCCCTGTAGGTGAGGTTGA-3′

139

mSOCS1-F mSOCS1-R

5′-CCGCTCCCACTCCGATTAC-3′ 5′-CGAAGAAGCAGTTCCGTTGG-3′

167

mSOCS3-F mSOCS3-R

5′-CCAGTCGGGGACCAAGAAC-3′ 5′-TGGGTGGCAAAGAAAAGGAG-3′

164

refer-actin

Tyro3

Axl

Mer

NF-κB

IκB

TLR-3

TLR-4

IL-1β

IL-6

TNF-α

IFN-β

SOCS1

SOCS3

primer sequence

primer size (bp)

14

endocrine disruptors in organisms.16 However, whether the reproductive toxicity in male animals induced by exposure to endocrine disruptor TiO2 NPs is involved in testitis as well as immunological dysfunction in the testis is unclear. Infection or noninfection and inflammation within the male reproductive system have detrimental effects on reproduction, which usually manifest as reduced androgen production, lowered sperm numbers, and temporary loss of fertility.31 Importantly, the testis presents a special immunological environment, considering its property of immune privilege that tolerates allo-antigens and autoantigens.31,33 Toll-like receptors (TLRs) are expressed in Sertoli cells of testis and can trigger testicular innate responses after activation by ligands. The TLR signaling pathway must be tightly controlled because unrestrained TLR activation generates a chronic

and motility and increased abnormal sperm and germ cell apoptosis in the testes.23,24 Our previous studies also demonstrated that exposure to anatase TiO2 NPs (5 nm) can migrate to the testis, leading to reductions in fertility, sperm concentration, sperm motility, luteinizing hormone, follicle stimulating hormone and testosterone, elevations of estradiol and progesterone, alterations of gene expression,25,26 and biochemical dysfunctions in the damaged testes of mice.27 TiO2 NPs exposure was suggested to significantly inhibit spermatogenesis and decrease the number of spermatogenic cells in mouse testes (NPs diameter = 100 nm)28 or in rat testes (NPs diameter = 21 nm).29 Recently, our study in vitro found that anatase TiO2 NPs (5 nm) could induce mitochondria-mediated or endoplasmic reticulum-mediated apoptotis of Sertoli cells from male mouse testes.30 Accordingly, TiO2 NPs may act as 347

DOI: 10.1021/acs.jafc.5b05262 J. Agric. Food Chem. 2016, 64, 346−355

Article

Journal of Agricultural and Food Chemistry

fixation, paraffin-embedded simples were sectioned at 5 μm, stained with hematoxylin and eosin (HE), and then routinely evaluated by a histopathologist unaware of the treatments for light microscopy (U-III Multipoint Sensor System; Nikon, Tokyo, Japan). Inflammatory Cells Analysis. After TiO2 NPs treatment in mice, mice were lightly anesthetized with ether, and the testes from five mice from each group were quickly dissected; suspensions containing inflammatory cells were isolated with the multiple digestion steps. Cells were collected by centrifugation and then washed twice with phosphate-buffered saline (PBS). An average of 90% of the total instilled PBS volume was retrieved both times, and the amounts did not differ among the groups. The resulting fluid was centrifuged at 400g for 10 min at 4 °C to separate the cells from the supernatant containing various surfactants and enzymes. The cell pellet was used for enumeration of total and differential cell counts as described by AshaRani et al.39 Macrophages, lymphocytes, neutrophils, and eosinophils recovered from the PBS solution were counted using dark field microscopy to assess the extent of phagocytosis. Assay of Cytokine Expression. Real-time reverse transcription PCR was performed in the Bio-Rad iQ5 detection system as previously described.40 In brief, RNA samples (10 μg) in the testes (n = 5 in each group) were reverse transcribed in a reaction volume of 30 μL of Promega reverse transcriptase reagent system. SYBR Green PCR amplification reagent (Qiagen) and gene-specific primers were used. Primer sets were used for real-time PCR assays and are exhibited in Table 1. The probes for Tyro3, Axl, Mer, TLR3, TLR 4, NF-κB, IκB, IL-lβ, IL-6, TNF-α, IFN-α, IFN-β, SOCS1, and SOCS3 in the testes were designed by the manufacturer and purchased from Shinegene Co. (Shanghai, China). The amount of DNA was normalized to the GAPDH and actin rRNA signals amplified in a separate reaction.41 Western Blot. Testicular tissues were homogenized in ice-cold RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.1% SDS], and protease inhibitor was added with the dose of 1% RIPA lysis buffer. Total protein (30−50 μg) was subjected to 8 or 12% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies: anti-Tyro3 (1:500), anti-Axl (1:1000), antiMer (1:500), anti-TLR3 (1:500), anti-TLR4 (1:500), anti-NF-κB (1:500), anti-IκB (1:500), anti-IL-lβ (1:500), anti-IL-6 (1:500), antiTNF-α (1:500), anti-IFN-α (1:500), anti-IFN-β (1:500), anti-SOCS1 (1:500), anti-SOCS3 (1:500), and anti-β-actin (1:2000). After three washings with TBST, the membranes were incubated with HRP conjugated secondary antibodies (1:10000) (Longshengtianshi Biotechnology Co., Suzhou, China) at room temperature for 2 h, followed by detection using enhanced chemiluminescence (ECL; Millipore, Bedford, MA, USA). Immunoreactive bands were visualized using Xray film. For quantitative analysis, bands were evaluated with ImageJ software, normalized for β-actin density. Statistical Analysis. Data are presented as the mean ± SD obtained from at least three separate experiments. Statistical analyses were performed using SPSS 19.0 software (Chicago, IL, USA), and statistical comparisons were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test. Differences were considered statistically significant when the P value was 12 g/kg BW after oral administration. In addition, the quantity of TiO2 NPs does not exceed 1% by weight of the food according to U.S. federal regulations. In addition, after 8 months, the fertility of 5 mg/kg BW of TiO2 NPs treated males (10) was tested by caging with control females (10) of proven fertility. Testicular Collection. After TiO2 NPs treatment in mice, mice were lightly anesthetized with ether, and the testes were quickly removed and placed on ice. Fresh testes from five mice in each group were quickly soaked in 4% paraformaldehyde solution for histopathological examination. The remaining fresh testes were quickly stored in liquid nitrogen and subsequently assayed for gene and protein expression. Histopathological Evaluation. The testes from five mice in each group were placed in 4% paraformaldehyde overnight at 4 °C. After

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RESULTS Fertility of Male Mice. Because male mice bearing fertility can be maintained for 6−8 months, we detected their fertility caused by exposure to TiO2 NPs for 8 consecutive months, as given in Figure 1. It can been observed that exposure to TiO2 NPs led to great decreases in the mating rate, pregnancy rate, and number giving birth/fetus, with reductions of 56.07, 58.79, and 58.41% (Figure 1, P < 0.05), respectively. Histopathological Evaluation. Histopathological changes of testes are displayed in Figure 2. As expected, control testis of mice showed normal architecture, neat array of cells, and full sperm, whereas TiO2 NP-exposed mice presented infiltration of inflammatory cells, spano-sperm or sperm breakages, rarefaction, apoptosis or necrosis of spermatogenic cells and Sertoli 348


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Figure 3. Numbers of inflammatory cells in the testes of mice following exposure to TiO2 NPs for 9 consecutive months. Different letters in the same parameter indicate significant differences between groups (P < 0.05). Values represent means ± SD (N = 5).

Figure 1. Effects of TiO2 NPs on conception of male mice following exposure to 5 mg/kg BW TiO2 NPs for 8 consecutive months. Different letters in the same parameter indicate significant differences between groups (P < 0.05). Values represent means ± SD (N = 10).

Gene Expression. To explore whether testicular inflammation in male mice induced by exposure to TiO2 NPs may be associated with TAM/TLR-mediated signal pathway, expression of Tyro3, Axl, Mer, TLR3, TLR 4, NF-κB, IκB, IL-lβ, IL-6, TNF-α, IFN-α, IFN-β, SOCS1, and SOCS3 genes was measured and is presented in Figure 5. With increasing TiO2 NPs dose, expression of Tyro3, Axl, Mer, IκB, SOCS1, and SOCS3 gened was significantly decreased in the TiO2 NPexposed testes in comparison to control, with reductions of 22.07, 44.6, and 61.97% for Tyro3, 19.03, 48.91, and 52.09% for Axl, 7.98, 48.93, and 59.04% for Mer, 13.68, 54.74, and 63.16% for IκB, 21.99, 29.0, and 37.9% for SOCS1, and 9.27, 55.21, and 64.86% for SOCS3, respectively; but expression of TLR3, TLR 4, NF-κB, IL-lβ, IL-6, TNF-α, IFN-α, and IFN-β genes was greatly elevated in the testes in a dose-dependent manner (Figure 4, P < 0.05), with increases of 30.0, 35.0, and 97.5% for TLR3, 37.0, 54.84, and 109.87% for TLR4, 22.55, 36.09, and 61.28% for NF-κB, 46.15, 92.31, and 123.08% for IL-lβ, 29.07, 69.77, and 81.98% for IL-6, 9.82, 79.47, and 88.39% for TNF-α, 35.78, 78.9, and 97.25% for IFN-α, and 10.19, 17.59, and 80.56% for IFN-β (Figure 4, P < 0.05), respectively. Western Blot Assay. To further confirm biotoxic effects of TiO2 NPs involved in the TAM/TLRs signal pathway, their protein expression was examined using Western blot and is shown in Figure 5. It can be observed that TiO2 NPs greatly reduced expression of Tyro3, Axl, Mer, IκB, SOCS1, and SOCS3 proteind, with decreases of 18.16, 32.03, and 66.6% for Tyro3, 14.7, 45.74, and 57.99% for Axl, 11.35, 49.58, and 72.62% for Mer, 11.25, 21.93, and 28.22% for IκB, 44.92, 50.27, and 73.8%% for SOCS1, and 8.11, 18.21, and 34.44% for SOCS3 (Figure 5, p < 0.05), respectively, and significantly induced expression of TLR3, NF-κB, IL-lβ, IL-6, TNF-α, IFNα, and IFN-β protein in the testes in a dose-dependent manner, with enhancements of 21.4, 65.54, and 156.03% for TLR3, 14.75, 61.31, and 69.34% for NF-κB, 8.05, 15.68, and 52.97% for IL-lβ, 2.54, 9.31, and 15.74% for IL-6, 6.95, 23.98, and 81.3% for TNF-α, 2.54, 9.31, and 15.74% for IFN-α, and 14.29, 25.83, and 41.7% for IFN-β (Figure 5, P < 0.05), respectively.

Figure 2. Histopathological observation of testicular tissue (400×) in male mice following exposure to TiO2 NPs for 9 consecutive months. The yellow oval indicates infiltration of inflammatory cells; the yellow arrow indicates vacuolation of seminiferous tubule; the black arrow indicates spano-sperm or breakages; the green arrow indicates spermatogenic cell and Sertoli cell apoptosis and or tissue necrosis; and the blue arrow indicates bleeding.

cells, and vacuolation of seminiferous tubule as well as bleeding in the testis (Figure 2). Inflammatory Cells. To further determine whether longterm TiO2 NPs exposure induces testicular inflammation, we analyzed inflammatory cell content in testis. As shown in Figure 3, the numbers of macrophages, lymphocytes, neutrophils, and eosinophils in the TiO2 NPs-exposed mice were obviously increased by 1.52-, 3.17-, and 5.91-fold, 1.36-, 3.18-, and 7-fold, 1.18-, 2.45-, and 5.27-fold, and 0.7-, 1.93-, and 3.19-fold (Figure 3, P < 0.05), respectively, indicating that TiO2 NPs exposure caused severe inflammation in testis.

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DISCUSSION Because of large-scale use of NMs and potential toxicity, their negative side effects on the reproductive system have received 349


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Figure 4. Effect of TiO2 NPs on mRNA expression involved in TAM/TLR3 signaling pathway in the testis of mice following exposure to TiO2 NPs for 9 consecutive months. Different letters in the same gene indicate significant differences between groups (P < 0.05). Values represent means ± SD (N = 5).

much attention,15,16,25−29,42,43 but reproductive toxicity involving immunological dysfunction caused by NMs as endocrine disruptors in the testes has received little attention. In the present study, immunological mechanisms of chronic testicular inflammation in mice induced by long-term exposure to endocrine disruptor TiO2 NPs were investigated. Previous research demonstrated that long-term exposure (for 2, 3, or 6 consecutive months) to TiO2 NPs resulted in titanium accumulation in the testes and thus decreased body weight, testis weight and relative testis weight, sperm concentration, and sperm motility and caused sperm lesion and significant pathological changes of testes and epididymis, but no inflammation in either organ.25−27 However, the present study found that long-term exposure (for 9 consecutive months) to TiO2 NPs resulted in low fertility (Figure 1) and infiltration of inflammatory cells in the testis (Figures 2 and 3). Furthermore, alterations of TAM/TLR3-mediated signal pathway-related gene and protein expression involving immunological function in mouse testitis were examined, suggesting that exposure to TiO2 NPs remarkably inhibited expression of Tyro3, Axl, Mer, IκB, SOCS1, and SOCS3 and induced expression of TLR3, TLR 4, NF-κB, IL-lβ, IL-6, TNF-α, IFN-α, and IFN-β in mouse testitis (Figures 4 and 5), which may be associated with reproductive toxicity, and are discussed below. As is well-known, the testis is an endocrine organ that is highly specialized to produce sperm and male sex hormones. According to morphology, it consists of two major compartments, the seminiferous tubules (site of spermatogenesis), which consist of Sertoli cells and germ cells, surrounded by the interstitium (site of testosterone production) that contains

Leydig cells, blood vessels, leukocytes, lymphatic vessels, and fibroblasts. After the establishment of systemic self-tolerance, advanced germ cells within the testis develop. Despite the expression of new surface and intracellular proteins by germ cells, these immunogenic autoantigens do not evoke an immunological attack, mainly due to the unique tolerogenic environment of the testis. Therefore, testis immune privilege is important for preventing detrimental immune responses against autoimmunogenic germ cells.44 In the testis, there is a high degree of permeability of the vascular endothelium, and substances within the circulation diffuse freely into the interstitium.45 Instead, the blood testis barrier/Sertoli cell barrier is located within the seminiferous tubules and includes the body of Sertoli cells and tight junctions formed between adjacent Sertoli cells. The blood testis barrier prevents leukocytes and antibodies from entering the seminiferous tubules and sequesters the majority of the autoantigenic germ cells from the immune system. The importance of the blood testis barrier in immune privilege and spermatogenesis was demonstrated by mice with Sertoli cell specific deletion of the androgen receptor.46 In the current study, exposure to TiO2 NPs resulted in infiltration or increased numbers of inflammatory cells (Figures 2 and 3), rarefaction, apoptosis or necrosis of germ cells and Sertoli cells, and vacuolation of seminiferous tubule in the testis (Figure 2), indicating impairment of the blood testis barrier/Sertoli cell barrier and immune or tolerogenic environment in mouse testitis. TLRs recognize specific bacterial and viral components and activate a downstream signaling cascade, which ultimately leads to the induction of inflammation. They (most commonly 2, 3, 350


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Figure 5. Effect of TiO2 NPs on protein expression involved in TAM/TLR3 signaling pathway in the testis of mice following exposure to TiO2 NPs for 9 consecutive months. Different letters in the same protein indicate significant differences between groups (P < 0.05). Values represent means ± SD (N = 5). 351


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impair immune balance in mouse testitis. It has been suggested that SOCS1 and SOCS3 protein can suppress both TLR and cytokine receptor signaling in immune cells,69 and regulate TAM inhibition by TLR signaling in Sertoli cells32 and dendritic cells.52 To further confirm whether TiO2 NPs may be associated with the TAM/TLR-mediated signal pathway in mouse testitis, we also evaluated alteration of SOCS1/SOCS3 expression, indicating that endocrine disruptor TiO2 NPs caused significant reduction in SOCS1/3 expression (Figures 4 and 5). These results suggest that suppression of SOCS1/3 expression may be associated with a reduction of TAM receptor expression in mouse testitis, which in turn resulted in exacerbation of testicular inflammation and reproductive toxicity in male mice. It has been suggested that TiO2 NPs site of accumulation and TiO2 NP-induced toxicity may not only relate to animal or organ type but also depend on the physicochemical properties of TiO2 NPs, such as specific surface area, crystal shape, zeta potential, and aggregate size.70−73 The surface charge or zeta potential of NP affects its aggregation in solution and its behavior in an electric or ionic field and determines its various effects in humans, rats, and mice.74−77 Importantly, the smaller the agglomerates, the more easily NPs will reach deeper zones in the organism and disperse in body fluids.78 The current study suggests that long-term consecutive exposure of anatase TiO2 NPs (5 nm) with low dose could cause reproductive toxicity and immunological dysfunction in mouse testitis, which may be associated with TiO2 NPs accumulation in mouse testes.25,26 As suggested, TiO2 NPs have been increasingly produced in the world and widely used in the food industry and agriculture, etc. Workers and consumers are unintentionally exposed to TiO2 NPs by oral consumption, inhalation, and dermal contact; therefore, the fertility reduction and the immunological dysfunction caused by TiO2 NPs uses should be of high concern, especially in human consumers or occupational workers. In summary, the present study showed that long-term exposure to endocrine disruptor TiO2 NPs exhibited low fertility and testicular inflammation in mice. Furthermore, the toxicity is demonstrated to involve numerous biomarkers of impairment of immune function in the testis; for example, endocrine disruptor TiO2 NPs suppressed expression of TAM receptors and SOCS1/3 and induced expression of TLRs and pro-inflammatory cytokines. Taken together, the reproductive toxicity and immunological dysfunction in male mice induced by long-term exposure to endocrine disruptor TiO2 NPs may be closely associated with dysfunction of TAM/TLR3-mediated signal pathway in mouse testitis.

and 4) have been demonstrated to express in mouse and rat testes.47−49 When the appropriate stimulus is provided, these TLRs are capable of being activated, inducing inflammation and oxidative stress.50 Our data suggested endocrine disruptor TiO2 NPs (stimulus) significantly induced TLR3 and TLR4 expression in mouse testis (Figures 4 and 5), which may be related to testitis generation in male mice (Figures 2 and 3). Importantly, some studies showed that Sertoli cells and Leydig cells can negatively regulate TLRs by Tyro3, Axl, and Mer (TAM) receptors and, hence, prevent prolonged inflammation and damage to the testis.32−34,51−54 TAM receptors have vital roles in regulating innate immunity via their inhibition of the inflammatory response and promotion of phagocytosis of apoptotic cells and membranous organelles by dendritic cells and macrophages51,52,55 and are constitutively expressed, and TLR3/4 expression is suppressed,32,35,36,51,53 thus triggering antiviral activity by inducing the production of inflammatory cytokines in Sertoli cells.32,34,36,54 In the current study, our findings showed that long-term exposure to endocrine disruptor TiO2 NPs greatly suppressed expression of Tyro3, Axl, and Mer, which in turn decreased negative regulation to TLR3/4 expression by TAM receptors in mouse testitis (Figures 4 and 5) and caused excessive innate immune response and testicular damages (Figure 2). As is well-known, NF-κB is a vital intracellular mediator of the inflammatory cascade. In quiescent cells, NF-κB is bound to inhibitory proteins (IκBs) that prevent NF-κB from migrating to the nucleus and locating in the cytoplasm. When an appropriate inducer (such as endocrine disruptor TiO2 NPs) affects the cell, IκBs are phosphorylated and degraded, leading to nuclear uptake of NF-κB and triggering gene transcription (the pro-inflammatory cytokines of IL-lβ, IL-6, TNF-α, IFN-α, and IFN-β).56 It has been suggested that the TLRs were regarded as one of the critical effectors in the innate immune systems.57,58 They bind their ligands and then induce activation of NF-κB and activator protein-1 (AP-1), finally promoting the expression of inflammation-related genes,58−61 thus conducting to bulk release of pro-inflammatory cytokines such as IL-1β, IL6, TNF-α, and IFNs62 and leading to testis injury. As expected, the present study found that the expression of NF-κB, IL-lβ, IL6, TNF-α, IFN-α, and IFN-β was significantly induced by exposure to TiO2 NPs, whereas the inhibitor of NF-κB activation, IκB expression, was inhibited (Figures 4 and 5), suggesting that decreased TAM expression caused high expression of TLR-driven inflammatory cytokines due to endocrine disruptor TiO2 NPs, which in turn exacerbated impairment of the immune environment in mouse testitis. Our previous studies have demonstrated that exposure to anatase TiO2 NPs (5 nm) could up-regulate expression of TLR2, TLR4, NF-κB, MIF, IL-lβ, IL-2, IL-6, TNF-α, IL-4, IL-10, IL18, TGF-β, and INF-γ and down-regulate IκB expression in the inflammatory liver, kidney, lung, and hippocampus of mice.63−67 As suggested, TLRs are widely expressed in immune cells and act as sensors of invading pathogens.35 Indeed, increased TLR expression can induce signaling cascades, resulting in high expression of immune and inflammatory factors for eliminating infection.35,36 On the other hand, excessive activation of TLR signaling would promote the pathogenesis of autoimmune and chronic inflammatory diseases.68 Accordingly, TLR signaling activation must be strictly modulated to maintain the immune balance.34−36 Consequently, excessive TLR3 and TLR4 expression caused by endocrine disruptor TiO2 NPs may

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AUTHOR INFORMATION

Corresponding Author

*(F.H.) Phone: 86-13915553751. E-mail: [email protected]. Author Contributions ∥

F.H., Y.W., and Y.Z. contributed equally to this work.

Funding

This work was supported by the National Natural Science Foundation of China (Grants 81473007, 81273036, 30901218), the Jiangsu University Brand Major Construction Project of Biotechnology Major of Huaiyin Normal University (PPZY2015A018), and the Bringing New Ideas Foundation of Huaiyin Normal University (201510323053X). 352


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Journal of Agricultural and Food Chemistry Notes

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The authors declare no competing financial interest.

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Exposure to TiO2 Nanoparticles Induces Immunological Dysfunction in

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