Titanium dioxide nanoparticle-induced testicular damage, spermatogenesis suppression, and gene expression alterations in male mice

Journal of Hazardous Materials 258–259 (2013) 133–143

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Titanium dioxide nanoparticle-induced testicular damage, spermatogenesis suppression, and gene expression alterations in male mice Guodong Gao a,1 , Yuguan Ze a,1 , Xiaoyang Zhao a,1 , Xuezi Sang a,1 , Lei Zheng a , Xiao Ze a , Suxin Gui a , Lei Sheng a , Qingqing Sun a , Jie Hong a , Xiaohong Yu a , Ling Wang a , Fashui Hong a,b,c,∗ , Xueguang Zhang b,c,∗∗ a

Medical College of Soochow University, Suzhou 215123, China Jiangsu Province Key Laboratory of Stem Cell Research, Soochow University, 708 Renmin Road, Suzhou 215007, China c Cultivation base of State Key Laboratory of Stem Cell and Biomaterials built together by Ministry of Science and Technology and Jiangsu Province, Suzhou 215007, China b

h i g h l i g h t s • • • • •

Exposure to TiO2 Exposure to TiO2 Exposure to TiO2 Exposure to TiO2 Exposure to TiO2

a r t i c l e

NPs could cross blood–testis barrier and be accumulated in testis. NPs caused testis and sperm lesions in male mice. NP decreased sperm numbers and sperm motility in male mice. NP resulted in imbalance of sex hormones in male mice. NP caused alteration of 142 genes expression of known function in testis.

i n f o

Article history: Received 28 December 2012 Received in revised form 21 April 2013 Accepted 27 April 2013 Available online 6 May 2013 Keywords: Titanium dioxide nanoparticles Testis damage Sperm lesion Sex hormones Gene expression profile

a b s t r a c t Although titanium dioxide nanoparticles (TiO2 NPs) have been demonstrated to accumulate in organs resulting in toxicity, there is currently only limited data regarding male reproductive toxicity by TiO2 NPs. In this study, testicular damage and alterations in gene expression profiles in male mice induced by intragastric administration of 2.5, 5, and 10 mg/kg body weight of TiO2 NPs for 90 consecutive days were examined. Our findings showed that TiO2 NPs can cross the blood–testis barrier to reach the testis and accumulate therein, which, in turn, results in testicular lesions, sperm malformations, and alterations in serum sex hormone levels. Furthermore, microarray analysis showed that 70 genes with known functions were up-regulated, while 72 were down-regulated in TiO2 NPs-exposed testes. Of the altered gene expressions, Ly6e, Adam3, Tdrd6, Spata19, Tnp2, and Prm1 are involved in spermatogenesis, whereas Sc4mol, Psmc3ip, Mvd, Srd5a2, Lep, and Cyp2e1 are associated with steroid and hormone metabolism. Hence, the production and application of TiO2 NPs should be carried out cautiously, especially by humans of reproductive age. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction With the widespread application of titanium dioxide nanoparticles (TiO2 NPs) in industry, medicine, daily life, waste-water

∗ Corresponding author at: Medical College of Soochow University, Suzhou 215123, China. Tel.: +86 0512 61117563; fax: +86 0512 65880103. ∗∗ Corresponding author at: Jiangsu Province Key Laboratory of Stem Cell Research, Soochow University, 708 Renmin Road, Suzhou 215007, China. E-mail addresses: Hongfsh [email protected] (F. Hong), [email protected] (X. Zhang). 1 These authors contributed equally to this work.

treatment, etc., numerous studies have unequivocally shown that TiO2 NPs exposure can migrate through different routes and accumulate in the kidney [1–3] and brain tissues of animals [4–10], which, in turn, can cause oxidative stress, inflammation, and apoptosis, ultimately resulting in organ injury and failure. Dysfunction of the kidney and brain may also affect the function of the reproductive system [11–13]; therefore, the potential risks to reproductive health should be investigated, especially in those who are occupationally exposed to TiO2 NPs. Komatsu et al. [14] reported that TiO2 NPs were taken-up by mouse Leydig cells in vitro and disrupted cellular viability and proliferation and dysregulated gene expression of heme oxygenase-1 (HO-1), a sensitive marker for oxidative stress, and steroidogenic

0304-3894/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.04.046

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acute regulatory protein, which regulates mitochondrial cholesterol transfer. TiO2 NPs exposure to follicles was shown to cause obvious morphological changes, decrease follicle survival and formation of antral follicles, and inhibit follicle development and oocyte maturation in vitro in a rat model [15]. Recently, we demonstrated that TiO2 NPs can translocate to the ovary resulting in ovarian injury, impaired fertility, and alterations of gene expression profiles in mice [16,17]. Furthermore, nanomaterials were also found to affect the male reproductive system. For example, male mice that were exposed to TiO2 NPs by intraperitoneal injection showed significantly decreased sperm numbers and motility, increased abnormal sperm in the epididymis and testis, and increased numbers of germ cell apoptosis in the testis without exhibiting significant testicular and/or epididymis lesions [18]. Exposure to black carbon nanoparticles was demonstrated to have adverse effects on male reproductive function in mice characterized by increased serum testosterone (T) levels and partial vacuolation of the seminiferous tubules [19]. Bai et al. [20] suggested that repeated intravenous injections of water-soluble multiwalled carbon nanotubes into male mice can cause reversible oxidative stress to the testis without affecting fertility. Further, amorphous nanosilica particles were confirmed to penetrate the blood–testis barrier and the nuclear membranes of spermatocytes, but did not produce any apparent testicular injuries [21]. Although previous studies have confirmed that TiO2 NPs can enter the reproductive system and induce injury, to date, however, none have examined whether TiO2 NPs induce testicular toxicity involving spermatogenesis damage and dysregulation of hormone production and/or gene expression. A recent review by Lan and Yang [22] confirmed that most nanomaterials induce adverse effects on spermatogenesis at various levels; however, the mechanisms underlying nanomaterial-induced disruption of spermatogenesis and penetration of the blood–testis barrier remain unclear. Thus, the aims of the present study were to test the hypothesis that TiO2 NPs exerts a toxic effect in the testis of mice by suppressing spermatogenesis and altering gene expression and to determine the mechanisms of TiO2 NPs toxicity in mouse testis. 2. Materials and methods 2.1. Chemical Anatase TiO2 NPs were prepared via controlled hydrolysis of titanium tetrabutoxide. Details of the synthesis and characterization of TiO2 NPs were described in our previous reports [7,23]. The average particle size of powdered TiO2 NPs suspended in 0.5% (w/v) hydroxypropylmethylcellulose (HPMC) K4 M (Sigma–Aldrich, St. Louis, MO, USA) solvent that the HPMC solution prepared with deionized and distilled water after 12 h and 24 h incubation was 5–6 nm and the surface area of the sample was 174.8 m2 /g. The mean hydrodynamic diameter of the TiO2 NPs in HPMC solvent was 294 nm (range, 208–330 nm) and the zeta potential after 12 and 24 h incubation was 7.57 and 9.28 mV, respectively [7].

Soochow University Institutional Animal Care and Use Committee and conformed to the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. An HPMC concentration of 0.5% was used as a suspending agent. TiO2 NP powder was dispersed onto the surface of 0.5% (w/v) HPMC, and then the suspending solutions containing TiO2 NPs were treated ultrasonically for 30 min and mechanically vibrated for 5 min. For the experiment, the mice were randomly divided into four groups (n = 30 each), including a control group (treated with 0.5%, w/v HPMC) and three experimental groups [2.5, 5, and 10 mg/kg body weight (BW) of TiO2 NPs]. About the dose selection in this study, we consulted the report of World Health Organization in 1969. According to the report, LD50 of TiO2 for rats is larger than 12,000 mg/kg BW after oral administration. In addition, the quantity of TiO2 NPs does not exceed 1% by weight of the food according to the Federal Regulations of US Government. They were equal to about 0.15–0.7 g TiO2 NPs of 60–70 kg body weight for humans with such exposure, which were relatively safe doses. The mice were weighed, volume of TiO2 NPs suspensions was calculated for each mouse, and the fresh TiO2 NPs suspensions were administered to the mice by intragastric administration every day for 90 days. Symptoms and/or mortality were observed and carefully recorded each day during the 90-day period. After 90 days, the mice were weighed, anesthetized using ether, and then sacrificed. Blood samples were collected by peri-orbital bleeding by quickly removing the eyeball. Serum was collected by centrifuging blood at 1200 × g for 10 min. The testes and epididymides were quickly removed, placed on ice, dissected, and then frozen at −80 ◦ C.

2.3. Titanium content analysis The frozen testicular tissues were thawed and approximately 50-mg samples were weighed, digested, and analyzed for titanium content. Briefly, prior to elemental analysis, the testicular tissues were digested overnight with nitric acid (ultrapure grade), combined with 0.5 mL of H2 O2 , and then incubated at 160 ◦ C in high-pressure reaction containers in an oven until the samples were completely digested. Then, the solutions were incubated at 120 ◦ C to remove any remaining nitric acid until the solutions were clear. Finally, the remaining solutions were diluted to 3 mL with 2% nitric acid. Inductively coupled plasma-mass spectrometry (ICPMS, Thermo Elemental X7; Thermo Electron Co., Waltham, MA, USA) was used to determine the titanium concentration in the samples. Indium (20 ng/mL) was chosen as an internal standard element. Elemental Ti (isotopes 48 Ti and/or 49 Ti) was then quantified using ICP-MS against Ti standards, which also contained the internal standard. The calculation of tissue TiO2 from Ti determinations was done as follows. First, the mass of Ti determined for a sample was divided by the atomic weight of Ti to obtain the number of moles of Ti in that sample. Next, to calculate the mass of TiO2 , the number of moles of Ti was multiplied by the molecular weight of TiO2 . The basis for the latter calculation is that the number of moles of Ti equals the number of moles of TiO2 .

2.2. Animals and treatment 2.4. Histopathological evaluation of testicular tissues One hundred and fifty CD-1 (Imprinting Control Region) male mice, aged 5 weeks with a mean body mass of 22 ± 2 g, were purchased from the Animal Center of Soochow University (Jiangsu, China) and housed in stainless steel cages in a ventilated animal care facility. The room temperature of the housing facility was maintained at 24 ± 2 ◦ C with a relative humidity of 60 ± 10% under a 12-h light/dark cycle. Distilled water and sterilized food were available ad libitum. Prior to dosing, the mice were acclimated to this environment for 5 days. All animal protocols were approved by the

All histopathological examinations were performed using standard laboratory procedures. Briefly, five sets of testicular tissues from 5 mice were embedded in paraffin blocks, sliced to 5-␮m thicknesses, and placed on separate glass slides (5 slices from each testis). After hematoxylin–eosin staining, the stained sections were evaluated by a histopathologist unaware of the treatments using an optical microscope (U-III Multi-point Sensor System; Nikon, Tokyo, Japan).

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2.5. Confocal Raman microscopy in testis sections

2.9. Enzyme-linked immunosorbent assay (ELISA) analysis

Raman analysis was performed using backscattering geometry in a confocal configuration at room temperature using a HR-800 Raman microscope system equipped with a 632.817 nm HeNe laser (JY Co. in Fort-de-France, Martinique). Reportedly, when the size of TiO2 NPs reached 6 nm, the Raman spectral peak was 148.7 cm−1 [24]. Laser power and resolution were approximately 20 mW and 0.3 cm−1 , respectively, while the integration time was adjusted to 1 s. The testicular specimens were embedded in paraffin blocks, sliced into 5-␮m thicknesses, and placed onto glass slides. The slides were dewaxed, hydrated, and then scanned using the confocal Raman microscope.

To determine Adam3, Axud1, Cfd, Cyp1b1, Cyp2e1, Gpx5, Gyk11, Lep, Ly6e, Prm1, Spata19, Tdrd6, Th, and Tnp2 levels in mouse testes tissues, commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) were employed to analyze each respective protein following the manufacturer’s instructions. The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash; Thermo Electron, Vantaa, Finland) and the concentrations of Adam3, Axud1, Cfd, Cyp1b1, Cyp2e1, Gpx5, Gyk11, Lep, Ly6e, Prm1, Spata19, Tdrd6, Th, and Tnp2 were calculated from a standard curve for each sample.

2.6. Sex hormone analysis Sera samples were evaluated for sex hormone content of estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), T, and sex hormone binding globulin (SHBG) using commercial kits (Bühlmann Laboratories AG, Schönenbuch, Switzerland). All biochemical assays were performed using a clinical automatic chemistry analyzer (Type 7170A; Hitachi, Tokyo, Japan).

2.7. Microarray analysis Gene expression profiles of testicular tissues isolated from 5 mice in the control and TiO2 NPs-treated groups were compared by microarray analysis using Illumina BeadChips purchased from Illumina, Inc. (San Diego, CA, USA). Total RNA was isolated using the Ambion Illumina RNA Amplification Kit (cat no. 1755) according to the manufacturer’s protocol, and stored at −80 ◦ C. RNA amplification is the standard method for preparing RNA samples for array analysis [25]. Total RNA was then submitted to Biostar Genechip Inc. (Shanghai, China) for RNA quality analysis using a BioAnalyzer, and cRNA was generated and labeled using the one-cycle target labeling method. cRNA from each mouse was hybridized for 18 h at 55 ◦ C on Illumina Human HT-12 v 3.0 BeadChips containing 45, 200 probes according to the manufacturer’s protocol and subsequently scanned using the Illumina BeadArray Reader 500 to identify differentially expressed genes and establish potential biological significance based on the Gene Ontology Consortium database (http://www.geneontology.org/GO.doc.html). Data analyses were performed with GenomeStudio software version 2009 (Illumina Inc.) by comparing all values obtained at each time point against the 0 h values. Data were normalized with the quantile normalization algorithm and genes were considered as detected if the detection p-value was <0.05. Statistical significance was calculated using the Illumina DiffScore, a proprietary algorithm that uses the bead standard deviation to build an error model. Only genes with a DiffScore ≤−13 and ≥13, corresponding to a p-value of 0.05, were considered statistically significant [26,27].

2.8. Quantitative real-time PCR (qRT-PCR) mRNA levels of Adam3, Axud1, Cfd, Cyp1b1, Cyp2e1, Gpx5, Gyk11, Lep, Ly6e, Prm1, Spata19, Tdrd6, Th, and Tnp2 in the mouse testes were determined using real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) [28]. Synthesized complimentary DNA was generated by qRT-PCR with primers designed with Primer Express Software (Applied Biosystems, Foster City, CA, USA) according to the software guidelines, and PCR primer sequences are listed Table 1.

2.10. Semen evaluation Each left cauda epididymis was dissected, incised with a pin in 1.5 mL of Dulbecco’s modified Eagle’s medium with 10 mg/mL bovine serum albumin, and incubated for several minutes to release the sperm. To assess sperm concentration, an optical microscopybased hemocytometer method was used with an appropriate counting chamber. For the sperm movement assessment, one drop of sperm suspension was placed on a microscope slide and the movement of 200 sperm cells was examined using a light microscope at 40× magnification. After hematoxylin–eosin staining, sperms were observed and photos were obtained using an optical microscope (U-III Multi-point Sensor System; Nikon). 2.11. Statistical analysis All results are expressed as mean ± standard error (SE). Significant differences were determined by Dunnett’s pair-wise multiple comparison t-test using SPSS 19 software (SPSS, Inc., Chicago, IL, USA). A p-value < 0.05 was considered statistically significant. 3. Results 3.1. Body and testicular masses and titanium content We calculated the net increase in body weight and testicular mass of the male mice treated with TiO2 NPs for 90 days. As shown in Table 2, with increased TiO2 NPs doses, the net increase in body weight, testicular, and testes masses were significantly reduced, and titanium accumulation was greatly increased (p < 0.05), whereas no titanium was detected in control tissues. 3.2. Histopathological evaluation Testis histopathological images are shown in Fig. 1. Unexposed testis appeared normal (Fig. 1), while testes from 5 mice of each group exposed to increased doses of TiO2 NPs exhibited severe pathological changes, including rare sperm, sperm breakages, rarefaction of Sertoli cell and androgone, Sertoli cell apoptosis, androgone necrosis of the seminiferous tubules, decreased germinative layer thickness, vacuolation, and irregular arrangement of Sertoli cells of the seminiferous tubules (Fig. 1). In addition, we also observed significant black agglomerates in the seminiferous tubules exposed to 10 mg/kg of TiO2 NPs (Fig. 1). Confocal Raman microscopy further showed a characteristic TiO2 peak in the black agglomerates (148 cm−1 ), which further confirmed the deposition of TiO2 in the testis (see spectrum B in the Raman insets in Fig. 1). 3.3. Semen evaluation After intragastric administration of TiO2 NPs for 90 consecutive days, we collected sperm from the cauda epididymis and examined the total sperm concentrations, sperm motility, and percentage of

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Fig. 1. Histopathological observations of testis of male mice following intragastric administration of TiO2 NPs for 90 consecutive days. The blue circle surrounds rare sperm or breakages, the yellow circle indicates cell rarefaction, the green circle shows androgen-induced necrosis, and the blue arrow indicates mesenchymal congestion. The green arrows show decreased germinative layer thickness, the red arrows indicate vacuolation, the yellow arrows exhibit irregular arrangement of Sertoli cells, and the red circle indicates black agglomerates in the testis. Arrow A indicates a representative Sertoli cell that did not engulf the TiO2 NPs, while arrow B denotes a seminiferous tubule loaded with TiO2 NPs. The right panels show the corresponding Raman spectra identifying the specific peaks at about 148 cm−1 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Real time PCR primer pairs. PCR primers used in the gene expression analysis. Gene name

Description

Primer sequence

Refer-actin

mactin-F mactin-R

5 -GAGACCTTCAACACCCCAGC-3 5 -ATGTCACGCACGATTTCCC-3

Primer size (bp)

Adam3

mAdam3-F mAdam3-R

5 -GGTAACGACAGCCAGCAGTAAT-3 5 -GCTTCTTGGTTGTGGTCTTCTT-3 

263 82



Prm1

mPrm1-F m Prm1-R

5 -CCGCCGCTCATACACCATAA-3 5 -TGGCGAGATGCTCTTGAAGTCT-3

107

Spata19

mSpata19-F mSpata19-R

5 -TGTCCAAAGAGTGTCCACCTCA-3 5 -CAAGGGCTCAGCGTTTAGAGT-3

81

Tdrd6

mTdrd6-F mTdrd65-R

5 -TTGCGTGTCTACTGAAATAGGTG-3 5 -ACTTTATTCAAATGTTGGGTTGC-3

81

Tnp2

mTnp2-F mTnp2-R

5 -GTAGCTCAGGGCGAAGATACAA-3 5 -TTCCTGTGACATCATCCCAAC-3

110

Lep

mLep-F mLep-R

5 -GCCACCTTGGTCACCTCATC-3 5 -GGAAGTTTCACAATCTGGGAAC-3 

92



Ly6e

mLy6e-F mLy6e-R

5 -GAGTCAGCGCCGAATCTTG-3 5 -AGGTCTTCTTTGCCCACCC-3

Axud1

mAxud1-F mAxud1-R

5 -CGCCCTTCATTAGCTGATGTT-3 5 -CAGAGCCTGCGTTTCTTGG-3

127 117





Cyp1b1

mCyp1b1-F mCyp1b1-R

5 -TTAGTATGCTTTTCGCTGTGACA-3 5 -GGGGAGTTATTCCTGGGTTATTA-3

81

Cyp2e1

mCyp2e1-F mCyp2e1-R

5 -CCTGCTGCCCATCATTATCC-3 5 -GCTCTTACCCACTGAGCCATCT-3

84





Gpx5

mGpx5-F mGpx5-R

5 -AGCAGATTGACTCGCACAGG-3 5 -CACTCATAAGCACTAGCTGACCC-3

Th

mTh-F mTh-R

5 -CCAAGCACTGAGTGCCATTAG-3 5 -GCCAGGAAAGGTTGGAGAAG-3 

99 

5 -ACGGCAAATGAAGTCAGAACA-3 5 -AAGACTACTAATGGGTCAGAAATGG-3

mCfd-F mCd74-R

Cfd

123

97

Table 2 Body and testicular weights, and titanium content in the mouse testes after intragastric administration of TiO2 NPs for 90 consecutive days. Index

TiO2 NPs (mg/kg BW)

Net increase of body weight (g) Testicular weight (g) Relative testicular weight (mg/g) Ti content (ng/g tissue)

0

2.5

5

19.65 ± 0.98a 0.28 ± 0.014a 7.67 ± 0.384a Not detected

15.55 0.25 7.56 82

± ± ± ±

0.78b 0.013b 0.378a 4.1a

10

11.76 0.21 7.25 171

± ± ± ±

0.59c 0.01c 0.36b 8.55b

8.21 0.17 6.59 320

± ± ± ±

0.41d 0.008d 0.329c 16c

Different letters indicate significant differences between groups (p < 0.05). Values represent mean ± SE (N = 10).

abnormal sperm. Statistically significant decreases in sperm numbers and sperm motility, and increased abnormal sperm in the TiO2 NPs-exposed group were observed with increased TiO2 NPs doses (Table 3, p < 0.01). As shown in Fig. 2, increased TiO2 NPs dosages induced toxicity-related changes in sperm morphology indicated by numerous significant breakages from the head to the tail (Fig. 2). 3.4. Sex hormone levels The effects of TiO2 NPs on serum hormones of male mice are shown in Table 4. Upon TiO2 NPs exposure, E2 and P4 levels were

significantly increased, whereas LH, FSH, and T were markedly decreased with increased dosages (p < 0.05). 3.5. Gene expression profile Treatment with a high dose (10 mg/kg BW) of TiO2 NPs resulted in the most severe testicular damage, which was used to detect gene expression profiles for further exploration of the mechanisms of TiO2 NPs-induced testicular damage. Messenger RNA from testicular tissues of vehicle control groups and 10 mg/kg BW TiO2 NPs-treated groups for 90 consecutive days were analyzed with

Table 3 Total sperm concentration, sperm motility, and percentage of abnormal sperm after intragastric administration of TiO2 NPs for 90 consecutive days. Index

TiO2 NPs (mg/kg BW)

6

Sperm concentration (×10 ) Motility rate of sperm (%) Percentage of abnormal sperm (%)

0

2.5

5

10

3.2 ± 0.16a 92 ± 4.6a 2 ± 0.1a

2.4 ± 0.12b 85 ± 4.25b 9 ± 0.45b

2.0 ± 0.1b 70 ± 3.5c 12 ± 0.6c

1.7 ± 0.085d 55 ± 2.75d 23 ± 1.15d

Different letters indicate significant differences between groups (p < 0.05). Values represent mean ± SE (N = 10).

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Fig. 2. Morphological changes in sperm from male mice following intragastric administration of TiO2 NPs for 90 consecutive days.

the Illumina BeadChips. The results showed that 254 genes were obviously altered in the TiO2 NPs-exposed testicular tissue compared with the control group. Of the altered genes, 153 were up-regulated and 101 down-regulated (Suppl Table S1). The gene expression profile of testicular tissue from the TiO2 NPs-treated mice was classified using the ontology-driven clustering approach of PANTHER analysis (https://panther.appliedbiosystems.com/), which categorized the 142 genes in the gene expression profile into 14 gene clusters, including molecule and ion binding, metabolism, signal transduction, response to oxidative stress, immune, growth

and development, spermatogenesis, transport, steroid and hormone metabolic processes, cytoskeleton, apoptosis, responses to wounding and stimulus, protein synthesis and processing, and transcription, whereas the function of 112 genes was unknown (Fig. 3). 3.6. qRT-PCR To verify the accuracy of the microarray assays, several genes with significantly different expression patterns were chosen for

Table 4 Changes of sex hormone levels of serum in mice after intragastric administration of TiO2 NPs for 90 consecutive days. Sex hormone level

TiO2 NPs (mg/kg BW) 0

E2 (pmol/L) P4 (nmol/L) LH (IU/L) FSH (IU/L) PRL (␮g/L) T (ng/dL) SHBG (nmol/L)

466 2.70 0.40 0.08 0.64 103 0.51

2.5 ± ± ± ± ± ± ±

23a 0.14a 0.02a 0.004a 0.032a 5.16a 0.026a

508 4.2 0.31 0.05 0.65 92 0.52

5 ± ± ± ± ± ± ±

25b 0.21b 0.016b 0.002b 0.032a 4.6b 0.026b

Different letters indicate significant differences between groups (p < 0.05). Values represent mean ± SE (N = 10).

576 7.1 0.24 0.02 0.63 81 0.51

10 ± ± ± ± ± ± ±

29c 0.36c 0.012c 0.001c 0.032a 4.05c 0.026c

687 10.05 0.14 0.01 0.64 73 0.53

± ± ± ± ± ± ±

34d 0.50d 0.007d 0.0005d 0.032a 3.67d 0.026d

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Fig. 3. Functional categorization of 254 genes. Genes were functionally classified based on the ontology-driven clustering approach of PANTHER.

further qRT-PCR analysis due to their association with spermatogenesis, steroid and hormone metabolic processes, apoptosis, oxidative stress, and signaling transduction. The Gpx5, Ly6e, and Th genes were up-regulated, whereas Adam3, Axud1, Cfd, Cyp1b1, Cyp2e1, Gyk11, Lep, Prm1, Spata19, Tdrd6, and Tnp were downregulated (Table 5). The qRT-PCR analysis of all genes displayed expression patterns comparable with the microarray data (i.e., either up- or down-regulation (Suppl Table S1)).

lower than those in unexposed mice. A scheme that links the TiO2 NPs and the changes in Adam3, Axud1, Cfd, Cyp1b1, Cyp2e1, Gyk11, Lep, Prm1, Spata19, Tdrd6, and Tnp regulation is depicted in Fig. 4.

4. Discussion The accumulation of TiO2 NPs is an important factor in reproductive toxicity evaluation in vivo, but it is not known how TiO2 NPs actually enter the testis. In this study, we examined the accumulation of TiO2 NPs in the testes after intragastric administration of TiO2 NPs for 90 consecutive days and showed that TiO2 NPs could cross the blood–testis barrier (Fig. 1d) and accumulate (Table 2), which in turn resulted in the reduction of relative testicular mass (Table 2), pathological changes in the testis (Fig. 1), and significantly decreased sperm concentrations, sperm motility, and increased abnormal sperm concentrations (Table 3) and/or sperm lesions in the cauda epididymis (Fig. 2). Guo et al. also observed that intraperitoneal injections with 200 and 500 mg/kg TiO2 NPs and/or ZnO

3.7. ELISA ELISA was performed to further confirm protein expression levels of Adam3, Axud1, Cfd, Cyp1b1, Cyp2e1, Gpx5, Gyk11, Lep, Ly6e, Prm1, Spata19, Tdrd6, Th, and Tnp2 in the mouse testicular tissues. As shown in Table 6, the expression levels of Gpx5, Ly6e, and Th were gradually elevated following exposure to 10 mg/kg TiO2 NPs, whereas the levels of Adam3, Axud1, Cfd, Cyp1b1, Cyp2e1, Gyk11, Lep, Prm1, Spata19, Tdrd6, and Tnp in TiO2 NPs-exposed mice were Table 5 Comparison of fold-difference between the control and 90 day 10 mg/kg BW dosage. Function

Gene

Ct

Fold

Chip data

Spermatogensis

Adam3 Prm1 Spata19 Tdrd6 Tnp2 Lep Ly6e

0.719092 0.894625 1.122797 0.841642 1.027322 2.023434 −2.921479

0.607479656 0.53788699 0.45920269 0.558008112 0.490620017 0.245972 7.576224071

0.4685322 0.5809125 0.5343887 0.3694942 0.3262624 0.2494796 4.72642

Apoptosis and oxidative stress

Axud1 Cyp1b1 Cyp2e1 Gpx5 Th

0.036466 0.22843 1.350747 −1.685039 −1.392357

0.975040466 0.853563269 0.39208898 3.215490885 2.625072012

0.6194346 0.6515008 0.30024 2.299376 3.213171

Signaling transduction

Cfd Gyk11

1.076616 0.999206

0.474139667 0.500275255

0.5777617 0.3333492

Values represent mean ± SE (N = 5).

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Table 6 Effects of TiO2 NPs on the levels of protein expression in mouse testis following intragastric administration of TiO2 NPs for consecutive 90 days. Protein expression (ng/mg tissue)

TiO2 NPs (mg/kg BW) 0

Adam3 Prm1 Spata19 Tdrd6 Tnp2 Lep Ly6e Axud1 Cyp1b1 Cyp2e1 Gpx5 Th Cfd Gyk11

563 415 891 689 739 683 655 758 318 477 371 301 808 598

10 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

28a 21a 37a 34a 44a 34a 16a 24a 33a 38a 18a 15a 40a 30a

273 189 274 272 322 156 4863 339 176 175 1443 853 317 299

± ± ± ± ± ± ± ± ± ± ± ± ± ±

14b 9b 14b 14b 16b 8b 243b 17b 9b 9b 72b 43b 16b 15b

Different letters indicate significant differences between groups (p < 0.05). Values represent mean ± SE (N = 5).

NPs for 7 days could greatly reduced sperm density and motility, increased sperm abnormality, and resulted in germ cell apoptosis in male mouse testis [18,29]. Intraperitoneal injections with 1.5 and 7.5 mg/kg SiO2 NPs for 35 days were demonstrated to decrease sperm count and the rate of sperm mobility, and to increase the rate of malformation significantly, in male rat testis [30]. NPs-induced testicular injury and spermatogenesis inhibition in male mice may be related to alterations in male sex hormone levels and testicular gene expression. Therefore, in the current study, we investigated

hormone levels in male mice and evaluated the regulation of all genes (n = ∼45,000) in the mouse testis, and found that the expression levels of 142 genes of known functions were significantly changed by long-term exposure to TiO2 NPs (Suppl Table S1). Suppl Table S1 shows categories of differentially expressed genes with known functions and the main results involving spermiogenesis and hormone levels are discussed hereinafter. Many genes are critical for spermatogenesis (e.g., Tnp1, Tnp2, Prm1, and Prm2) and are expressed in a germ cell type-specific manner and/or a seminiferous epithelial stage-specific manner [31]. Spermiogenesis requires extensive chromatin condensation, which is dependent on histone displacement, a process in which histones are initially replaced by Tnp1 and Tnp2 proteins and subsequently by Prm1 and Prm2. Genetic ablation of transition proteins or protamines causes defective spermiogenesis [32–38]. Interestingly, our data showed that genes related to spermatogenesis were down-regulated (Suppl Table S1); for example, Prm1 and Tnp2 were significantly suppressed with DiffScores of −35.2756 and −32.004 under TiO2 NPs-induced toxicity, respectively. Decreased expressions of Prm1 and Tnp2 were further confirmed by qRT-PCR and ELISA assays (Tables 5 and 6). Reportedly, Adam3, Spata19, and Tdrd6 play critical roles in the process of spermatogenesis, in which Adam1a/Adam2 has been found to have a chaperone activity, serving to direct Adam3 to the sperm membrane [37,38]. Other studies have demonstrated that ablation of the Adam3 gene impaired sperm–zona pellucida binding [39], as Adam3 acts downstream of the Adam network in sperm assembly and functions in sperm–zona pellucida binding [40]. Spata19 is a testis-specific protein, which contains a mitochondria-targeting signal and works as an adhesive molecule between the adjacent mitochondria of

Fig. 4. A schematic showing possible mechanisms of TiO2 NPs induced damages of mouse testis.

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the sheath, and has an important role in spermiogenesis [41–43]. The Tdrd gene maternally functions in pole cells and in abdominal formation, and participates in the localization of mitochondrial RNAs to polar granules [44–46]. The present study found that Adam3, Spata19, and Tdrd6 were obviously suppressed, as indicated by DiffScores of −19.3334, −24.2952, and −19.8273 under TiO2 NPs-induced toxicity (Suppl Table S1), respectively. qRT-PCR and ELISA assays also suggested TiO2 NPs exposure resulted in reductions of Adam3, Spata19, and Tdrd6 expressions in mouse testis (Tables 5 and 6). Additionally, we found that TiO2 NPs exposure significantly induced Ly6e expression with a DiffScore of 139.6373 (Suppl Table S1). Increased expression of Ly6e was further confirmed by qRT-PCR and ELISA assays (Tables 5 and 6). However, LY6e expression was demonstrated to have no influence on testicular development or spermatogenesis [47]. Therefore, decreased Prm1, Tnp2, Adam3, Spata19, and Tdrd6 expression by TiO2 NPs exposure may induce reduction in sperm density in the epididymis and inhibit spermatogenesis in the testis. Male sex hormones are key to spermatogenesis. In the current experiment, the TiO2 NPs-exposed male mice had significantly elevated concentrations of serum E2 and P4, combined with greatly reduced amounts of T, LH, and FSH (Table 4). In fact, E2 has been shown to directly affect steroidogenesis in the rat testis via accumulation of estrogen-regulated proteins [48] and E2 excess stimulates Leydig cell hyperplasia in rodents and has been associated with cryptorchidism, testicular cancer, and impaired spermatogenesis [49]. In addition, E2 plays a critical role in regulating gonadotropin secretion in both male rodents and humans. FSH and LH are glycoprotein hormones secreted by the anterior pituitary and act directly on the testis to stimulate somatic cellular functions in support of spermatogenesis. Studies have also demonstrated that circulating LH and FSH concentrations were effectively reduced by E2 [50,51]. In the present study, our data suggested that consecutive exposure to TiO2 NPs effectively increased E2 and significantly decreased LH and FSH levels in male mice, which severely suppressed spermatogenesis. However, the mechanism behind increased P4 levels following TiO2 NPs exposure is unknown. But, since progesterone is a precursor of T, TiO2 NPs-induced T reduction might explain this result because T is produced in the interstitial section of testis under the influence of LH secreted from the pituitary gland [52]. It is also believed that FSH increases Sertoli cell synthesis of an androgen binding protein required for maintenance of high T concentrations [53]. Therefore, the reduction of LH and FSH released from the pituitary gland inhibited T production by TiO2 NPs exposure, which was associated with Sertoli cell damage (Figs. 1 and 2). Our findings showed that sex hormone balance in the male reproductive system was disrupted by TiO2 NPs exposure and thereby suppressed spermatogenesis through pathological changes in the testis. However, further studies will be required to fully elucidate the nature of the possible direct roles of E2 and P4 on testicular and epididymal function. Indeed, differential expression of genes with known functions involved in steroid and hormone metabolic processes were observed (Suppl Table S1). For example, Sc4mol, Mvd, and Srd5a2 were up-regulated by 90.91438-, 14.808-, and 14.31332fold, whereas Cyp2e1 and Lep were down-regulated by −44.2442and −40.8036-fold, respectively, under TiO2 NPs-induced toxicity. The reductions of Cyp2e1 and Lep expressions caused by TiO2 NPs exposure were also confirmed by qRT-PCR and ELISA assays (Tables 5 and 6). Sc4mol is a member of the cholesterol biosynthesis pathway and catalyses the demethylation of C4-methylsterols, and its deficiency leads to inborn errors of cholesterol biosynthesis [54]. It was suggested that increased Sc4mol and Mvd expression disrupted steroid hormone biosynthesis and metabolism in the cryptorchid testis and resulted in decreased T levels [55,56]. Reportedly, Srd5a2 activity plays a key role in androgen-regulated reproductive mechanisms. In the Srd5a2 gene promoter region,

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progesterone and androgen response elements were identified in mice, suggesting that different androgens might be able to regulate Srd5a2 gene expression [57]. T can regulate the enzymatic activity of 5-a reductase in a system [58] and Srd5a2 mRNA expression in prostates of intact and castrated rats in vivo [59,60]. In the present study, elevated Srd5a2 expression accompanying increased P and decreased T levels was observed following long-term exposure to TiO2 NPs. Cytochrome P450 2E1 (Cyp2e1) participates in the oxidative metabolic processes of steroid hormones, drugs, triglycerides, and xenobiotics [61]. Hence, decreased Cyp2e1 expression can decrease steroid hormone metabolism following TiO2 NPs exposure. Leptin (LeP) plays a specific role in mediating the response of reproductive hormones to the nutritional status of the organism and prevents fasting-induced reductions in serum T and LH levels [62]. Therefore, Lep down-regulation also resulted in reduction of serum T and LH levels in male mice following exposure to TiO2 NPs. The Leydig cells secrete T, an essential hormone for spermatogenesis. Komatsu et al. [14] exposed the mouse Leydig cell line TM3 to 25–70-nm diesel exhaust particles and 14-nm black carbon NPs and observed significantly increased HO-1 expression levels, which is a sensitive marker for oxidative stress, and steroidogenic acute regulatory (StAR) in TM3 cells, which is an important protein in T biosynthesis. Rats exposed to air containing nanomaterialrich diesel exhaust (NR-DE) for 1 and 2 months showed increased plasma T levels and up-regulated StAR, cytochrome P450 side chain cleavage, growth hormone receptor, and insulin-like growth factor1 (IGF-1) mRNA expression levels. The disruption of T biosynthesis caused by NR-DE exposure may be explained by molecular changes in StAR and P450 levels via growth hormone signaling [63]. 5. Conclusion The present study suggests that TiO2 NPs can enter the testis via the blood–testis barrier and accumulate in the mouse testis, which in turn can cause testicular damage, sperm lesions, and decreased spermatogenesis in the cauda epididymis. Furthermore, testicular dysfunctions following exposure to TiO2 NPs may be primarily related to significant alterations in the expression of genes involved in spermatogenesis, as well as steroid and hormone metabolic processes, etc. Our findings imply that long-period exposure of low dose TiO2 NPs should be of concern. Therefore, the application of TiO2 NPs in various areas and their production by male workers should be monitored carefully. Acknowledgements This work was supported by the National Natural Science Foundation of China (grant nos. 81273036, 30901218), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2013.04.046. References [1] J.F. Zhao, J. Wang, S.S. Wang, X.Y. Zhao, J.Y. Yan, J. Ruan, N. Li, Y.M. Duan, H. Wang, F.S. Hong, The mechanism of oxidative damage in nephrotoxicity of mice caused by nano-anatase TiO2 , J. Exp. Nanosci. 5 (5) (2010) 447–462. [2] S.X. Gui, Z.L. Zhang, L. Zheng, Q.Q. Sun, X.Z. Sang, X.R. Liu, G.D. Gao, Y.L. Cui, Z. Cheng, J. Cheng, M. Tang, F.S. Hong, The molecular mechanism of kidney injury of mice caused by exposure to titanium dioxide nanoparticles, J. Hazard. Mater. 195 (2011) 365–370. [3] S.X. Gui, X.Z. Sang, L. Zheng, Y.G. Ze, X.Y. Zhao, L. Sheng, Q.Q. Sun, Z. Cheng, J. Cheng, R.P. Hu, L. Wang, M. Tang, F.S. Hong, Intragastric exposure to titanium

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