Titanium dioxide nanoparticles oral exposure induce osteoblast apoptosis, inhibit osteogenic ability and increase lipogenesis in mouse.

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in food, paint, coating, cosmetic, and composite orthodontic material. As a common food additive, TiO2-NPs can accumulate in various organs of human body, but the effect and underlying mechanism of bone remain unclear. Here mice were exposed to TiO2-NPs by oral gavage, and histological staining of femoral sections showed that TiO2-NPs reduced bone formation and enhanced osteoclast activity and lipogenesis, contributing to decreased trabecula bone. Transmission electron microscope (TEM) as well as biochemical and flow cytometry analysis of osteoblast exhibited that TiO2-NPs accumulated in osteoblast cytoplasm and impaired mitochondria ultrastructure with increased reactive oxygen species (ROS) and lipid hyperoxide, resulting in osteoblast apoptosis. In terms of mechanism, TiO2-NPs treatment inhibited expression of AKT and then increased pro-apoptotic protein Bax expression which was failure to form heterodimers with decreased anti-apoptotic Bcl-2, activating downstream Caspase-9 and Caspase-3 and inducing apoptosis. Additionally, TiO2-NPs suppressed Wnt3a level and then activated anti-Glycogen synthesis kinase (GSK-3ß) phosphorylation, and ultimately resulted in degradation of ß-catenin which down-regulated Runt-related transcription factor 2 (Runx2) and Osterix, inhibiting expression of osteogenic related proteins. Together, these results revealed that exposure of TiO2-NPs induced apoptosis and inhibited osteoblast differentiation through suppressing PI3K/AKT and Wnt/ß-catenin signaling pathways, resulting in reduction of trabecula bone.

Impairment of ovarian follicular development caused by titanium dioxide nanoparticles exposure involved in the TGF-ß/BMP/Smad pathway.

Titanium dioxide nanoparticles (TiO2 NPs) have been shown to induce reproductive system damages in animals. To better underline how TiO2 NPs act in reproductive system, female mice were exposed to 2.5, 5, or 10 mg/kg TiO2 NPs by gavage administration for 60 days, the ovary injuries, follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels as well as ovarian follicular development-related molecule expression were investigated. The results showed that TiO2 NPs exposure resulted in reduction of ovary weight and inhibition of ovarian follicular development. Furthermore, the suppression of follicular development was demonstrated to be closely related to higher FSH and LH levels, and higher expression of activin, follistatin, BMP2, BMP4, TGF-ß1, Smad2, Smad3, and Smad4 as well as decreased inhibin-α expression in mouse ovary in a dose-dependent manner. It implies that the impairment of ovarian follicular development caused by TiO2 NPs exposure may be mediated by TGF-ß signal pathway.

Damage to the Blood Brain Barrier Structure and Function from Nano Titanium Dioxide Exposure Involves the Destruction of Key Tight Junction Proteins in the Mouse Brain.

Numerous studies have proven that nano titanium dioxide (nano TiO2) can accumulate in animal brains, where it damages the blood brain barrier (BBB); however, whether this process involves destruction of tight junction proteins in the mouse brain has not been adequately investigated. In this study, mice were exposed to nano TiO2 for 30 consecutive days, and then we used transmission electron microscopy to observe the BBB ultrastructure and the Evans blue assay to evaluate the permeability of the BBB. Our data suggested that nano TiO2 damaged the BBB ultrastructure and increased BBB permeability. Furthermore, we used immunofluorescence and Western blotting to examine the expression of key tight junction proteins, including Occludin, ZO-1, and Claudin-5 in the mouse brain. Our data showed that nano TiO2 reduced Occludin, ZO-1 and Claudin-5 expression. Taken together, nano TiO2-induced damage to the BBB structure and function may involve the destruction of key tight junction proteins.

Nano-TiO2 Reduces Testosterone Production in Primary Cultured Leydig Cells from Rat Testis Through the Cyclic Adenosine Phosphate/Cyclic Guanosine Phosphate/Epidermal Growth Factor Receptor/Matrix Metalloproteinase Signaling Pathway.

Nano-titanium dioxide (nano-TiO2) has been shown to inhibit testosterone synthesis in male mice or rats; however, the mechanisms underlying these effects have yet to be elucidated. In this study, we investigated whether the inhibition of testosterone synthesis by nano-TiO2 on Leydig cells (LCs) was related to the dysfunction of the cAMP/CGMP/EGFR/MMP signaling pathway in primary cultures of LCs prepared from rat testis exposed to nano-TiO2. We found that the early apoptotic rate of LCs increased by 4.34 and 4.94 times, respectively, after exposure to 20 g/mL and 40 g/mL nano-TiO2 ; we also found that NO increased by 1.1 and 2.86 times, respectively. ROS increased by times of 0.71, 3.15 and 3.43; RNS increased by 0.62, 1.34 and 1.14 times; and SOD activity decreased by 18.3%, 28.16%, and 67.6%, respectively, when the concentration of nano-TiO2 was 10, 20 and 40 g/mL. These results indicated that nano-TiO2 treatment resulted caused damage to the LCs, including an imbalance of oxidation and antioxidation. Following nano-TiO2 treatment, the cAMP content had decreased by 48%, 48% and 47.6%; cGMP content had decreased by 18.7%, 52.2% and 56.7%; the levels of ATP in the LCs had decreased by 15.15%, 45.75% and 66.67%; the expression of HCGR protein had decreased by 26.7%, 45.07% and 74.64%; the expression of LHR protein had decreased by 18.3%, 28.16% and 67.6%; and the levels of T had decreased by 34.48%, 46.62% and 44.12%. Collectively, our results indicated that the inhibition of testosterone production by nano-TiO2 is related to the dysfunction of the cAMP/CGMP/EGFR/MMP signaling pathway.

Titanium Dioxide Inhibits Hippocampal Neuronal Synapse Growth Through the Brain-Derived Neurotrophic Factor-Tyrosine Kinase Receptor B Signaling Pathway.

Nanoparticulate titanium dioxide (nano-TiO2) is a commonly used nanoparticle material and has been widely used in the fields of medicine, cosmetics, construction, and environmental protection. Numerous studies have demonstrated that nano-TiO2 has toxic effects on neuronal development, which lead to defects in learning and memory functions. However, it is still unclear whether nano-TiO2 inhibits the development of synapse and the underlying molecular mechanism is still unknown. In this study, nano-TiO2 was administered to rat primary hippocampal neurons for 24 h to investigate the underlying molecular mechanisms behind the inhibition of neuronal synaptic development by nano-TiO2. We used hippocampal neurons as a model to study the effect of nano-TiO2 on synaptic development. Our results demonstrated that dendritic development that represented synaptic plasticity in hippocampal neurons was significantly inhibited in a concentration-dependent manner after exposure to nano-TiO2 for 24 h. Experiments with varying concentrations of nano-TiO2 (5, 15, and 30 g/mL) indicated that the apoptotic rate of hippocampal neurons increased, development of neuronal synapses were inhibited, and synaptic densities decreased by 24.29%, 54.29%, and 72.86%, respectively, in post-treatment with nano-TiO2. Furthermore, the results indicated that the expressions of Synapsin I (SYN I) and postsynaptic density 95 (PSD95) in neuron synapse were also significantly inhibited, particularly SYN I decreased by 18.43%, 37.2%, and 51.6%, and PSD95 decreased by 16.02%, 24.06%, and 38.74% after treatment with varying concentrations of nano-TiO2, respectively. In addition, experiments to assess the BDNF-TrkB signaling pathway indicated that nano-TiO2 inhibited the expressions of key proteins in the downstream MEK/ERK and PI3K/Akt signaling pathways by inhibiting the expression of BDNF. With concentrations of nano-TiO2 at 5, 15, and 30 µg/mL, the expression of BDNF decreased by 22.64%, 33.3%, and 53.58% compared with the control group. Further, the expression ratios of downstream key proteins p-CREB/CREB decreased by 3.03%, 18.11%, and 30.57%; p-ERK1/2/ERK1/2 ratios decreased by 19.11%, 28.82%, and 58.09%, and p-Akt1/Akt1 ratios decreased by 1.92%, 27.79%, and 41.33%, respectively. These results demonstrated that nano-TiO2 inhibited the normal function of the BDNF-TrkB signaling pathway, which is closely related to neuronal synapse. Thus, it can be hypothesized that the inhibition of neuronal synaptic growth by nano-TiO2 may be related to the inhibition of BDNF-TrkB signaling pathway.

Molecular mechanism of mice gastric oxidative damage induced by nanoparticulate titanium dioxide.

BACKGROUND: Nanoparticulate titanium dioxide (Nano-TiO2) has been widely used in food industry, and it has been demonstrated to have adverse effects on mice and human stomach, but its mechanism is rarely concerned. The aim of this study is to determine the effects of nano-TiO2 on the stomach and confirm the role of oxidative stress and apoptosis in the mice gastric damage caused by nano-TiO2, as well as its molecular mechanisms. METHODS: Mice were continuously exposed to nano-TiO2 with 1.25, 2.5 and 5 mg/kg bw by intragastric administration for 9 months in the present study. The ultrastructure, levels of reactive oxygen species (ROS) and peroxides, activities of antioxidant enzymes and mitochondria-related enzymes, ATP contents as well as apoptosis-related factors expression in mice stomach were examined. RESULTS: Oxidative stress, apoptosis and nano-TiO2 aggregation were found in gastric mucosal smooth muscle cells after nano-TiO2 exposure. Nano-TiO2 exposure also resulted in the over-production of ROS and peroxides, decrease of ATP production and activities of antioxidant enzymes and mitochondria-related ATPases, upregulation of apoptosis-related factors including γH2AX, Cyt c, caspase 3, and p-JNK expression, and down-regulation of Bcl-2 expression in mice stomach. CONCLUSIONS: The gastric toxicity of mice induced by chronic exposure to low dose nano-TiO2 may be associated with oxidative stress and mitochondria-mediated apoptosis in mice.

Liver Inflammation and Fibrosis Induced by Long-Term Exposure to Nano Titanium Dioxide (TiO2) Nanoparticles in Mice and Its Molecular Mechanism.

Titanium dioxide (TiO2) and nano-sized titanium dioxide (nano-TiO2), which are used in food production, may be harmful to the body. Long-term exposure to nano-TiO2 can lead to hepatic injury; however, the effect of nano-TiO2 on liver fibrosis and the underlying mechanism remain unclear. The TGF-ß/Smad/MAPK/Wnt signaling pathway is important for tissue fibrosis. In this study, mice were fed nano-TiO2 (2.5, 5, and 10 mg/kg body weight) for nine consecutive months to investigate its effect on liver fibrosis. Nano-TiO2 induced hepatic inflammatory cell infiltration and hepatic fibrosis and upregulated the expression of HIF-1α (+75-fold to +2.38-fold), Wnt3 (+12% to +135%), Wnt4 (1.33-fold to 6-fold), NF-κB (+3.13% to +34.38%), TGF-ß1 (+1307-fold to +1.85-fold), TGF-ß1R (+0.8-fold to 1.33-fold), Smad-2 (+0.58-fold to +1.58-fold), ILK (+0.43-fold to +1.19-fold), ECM (+1.82-fold to 2.36-fold), calpain 2 (+0.11-fold to +0.78-fold), α-SMA (+0.63-fold to +1.56-fold), c-Myc (+0.27-fold to +0.46-fold), and collagen I (+8% to +36%), and increased the phosphorylation level of p38MAPK (+66.67% to +153.33%) in inflammatory and fibrotic liver tissues, whereas it downregulated cyclin D (-6.25% to -43.75%) and decreased the phosphorylation levels of GSK-3ß (-3.12% to -46.88%) and ß-catenin (-19.57% to -45.65%). These results indicate that hepatic fibrosis induced by nano-TiO2 is mediated by the TGF-ß/Smads/MAPK/Wnt signaling pathway. This study provides insight into the mechanism underlying hepatic toxicity induced by nano-TiO2 .

Molecular mechanism of nanoparticulate TiO2 induction of axonal development inhibition in rat primary cultured hippocampal neurons.

Numerous studies have demonstrated the in vitro and in vivo neurotoxicity of nanoparticulate titanium dioxide (nano-TiO2 ), a mass-produced material for a large number of commercial and industrial applications. The mechanism of nano-TiO2 -induced inhibition of axonal development, however, is still unclear. In our study, primary cultured hippocampal neurons of 24-hour-old fetal Sprague-Dawley rats were exposed to 5, 15, or 30 µg/mL nano-TiO2 for 6, 12, and 24 hours, and the toxic effects of nano-TiO2 exposure on the axons development were detected and its molecular mechanism investigated. Nano-TiO2 accumulated in hippocampal neurons and inhibited the development of axons as nano-TiO2 concentrations increased. Increasing time in culture resulted in decreasing axon length by 32.5%, 36.6%, and 53.8% at 6 hours, by 49.4%, 53.8%, and 69.5% at 12 hours, and by 44.5%, 58.2%, and 63.6% at 24 hours, for 5, 15, and 30 µg/mL nano-TiO2 , respectively. Furthermore, nano-TiO2 downregulated expression of Netrin-1, growth-associated protein-43, and Neuropilin-1, and promoted an increase of semaphorin type 3A and Nogo-A. These studies suggest that nano-TiO2 inhibited axonal development in rat primary cultured hippocampal neurons and this phenomenon is related to changes in the expression of axon growth-related factors.

Wnt Pathway-Mediated Nano TiO2-Induced Toxic Effects on Rat Primary Cultured Sertoli Cells.

Nanosized titanium dioxide (Nano TiO2) has been widely used in daily lives, medicine, industry, and caused the potential reproduction toxicity for animals and human, however, the underlying molecular mechanisms for the reproductive toxicity of nano TiO2 are still largely unclear. In the present study, when primary cultured rat Sertoli cells (SCs) were exposed to nano TiO2, cell injury and alterations in wingless related MMTV integration site (Wnt) pathway-related factors including Wnt1, Wnt3a, Wnt5a, Wnt11, ß-catenin, and p-GSK-3ß expression were investigated. The results suggested that nano TiO2 could be translocated to cytoplasm or nucleus, and decreased cell viability, and impaired morphological structures of SCs, induced apoptosis and dead of primary cultured rat SCs. Furthermore, nano TiO2-induced the toxicity of primary cultured rat SCs was associated with increased expression of Wnt1, Wnt3a, Wnt5a, Wnt11, and ß-catenin and involved with reduced p-GSK-3ß expression. Therefore, this implies that nano TiO2-induced toxic effects on SCs may be associated with Wnt signaling pathways.

Suppression of testosterone production by nanoparticulate TiO2 is associated with ERK1/2-PKA-PKC signaling pathways in rat primary cultured Leydig cells.

BACKGROUND: Nanoparticulate titanium dioxide (nano-TiO2) enters the body through various routes and causes organ damage. Exposure to nano-TiO2 is reported to cause testicular injury in mice or rats and decrease testosterone synthesis, sperm number, and motility. Importantly, nano-TiO2 suppresses testosterone production by Leydig cells (LCs) and impairs the reproductive capacity of animals. METHODS: In an attempt to establish the molecular mechanisms underlying the inhibitory effect of nano-TiO2 on testosterone synthesis, primary cultured rat LCs were exposed to varying concentrations of nano-TiO2 (0, 10, 20, and 40 µg/mL) for 24 hours, and alterations in cell viability, cell injury, testosterone production, testosterone-related factors (StAR, 3ßHSD, P450scc, SR-BI, and DAX1), and signaling molecules (ERK1/2, PKA, and PKC) were investigated. RESULTS: The data show that nano-TiO2 crosses the membrane into the cytoplasm or nucleus, triggering cellular vacuolization and nuclear condensation. LC viability decreased in a time-dependent manner at the same nano-TiO2 concentration, nano-TiO2 treatment (10, 20, and 40 µg/mL) decreased MMP (36.13%, 45.26%, and 79.63%), testosterone levels (11.40% and 44.93%), StAR (14.7%, 44.11%, and 72.05%), 3ßHSD (26.56%, 50%, and 79.69%), pERK1/2 (27.83%, 63.61%, and 78.89%), PKA (47.26%, 70.54%, and 85.61%), PKC (30%, 50%, and 71%), SR-BI (16.41%, 41.79%, and 67.16%), and P450scc (39.41%, 55.26%, and 86.84%), and upregulated DAX1 (1.31-, 1.63-, and 3.18-fold) in primary cultured rat LCs. CONCLUSION: Our collective findings indicated that nano-TiO2-mediated suppression of testosterone in LCs was associated with regulation of ERK1/2-PKA-PKC signaling pathways.

Nanoparticulate TiO2 -mediated inhibition of the Wnt signaling pathway causes dendritic development disorder in cultured rat hippocampal neurons.

Titanium dioxide nanoparticles (TiO2 NPs) are increasingly used in daily life, in industry, and in environmental clearing, but their potential neurodevelopmental toxicity has been highly debated. In this study, we explored whether TiO2 NPs inhibited development of dendritic morphology and identified possible molecular mechanisms associated with this inhibition in primary cultured rat hippocampal neurons. Results showed that TiO2 NPs decreased neurite length, the number of branches and the spine density, and impaired mitochondrial function in the developing neurons. Furthermore, TiO2 NPs significantly reduced the expression of several proteins involved in canonical Wnt3a/ß-catenin signaling including Wnt3a, ß-catenin, p-GSK-3ß, and CyclinD1 and conversely, elevated GSK-3ß expression. In addition to altering expression of proteins involved in canonical Wnt3a/ß-catenin signaling, TiO2 NPs decreased expression of proteins invovled in non-canonical Wnt signaling, including, MKLP1, CRMP3, ErbB4, and KIF17. Taken together, these results indicate that suppression of dendritic development caused by TiO2 NPs is associated with inhibition of activation of the Wnt/ß-catenin pathway or non-canonical Wnt pathway-induced expression of microtubule cytoskeletal components in the developing neurons. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2139-2149, 2017.

Toxic effects of TiO2 nanoparticles in primary cultured rat sertoli cells are mediated via a dysregulated Ca2+ /PKC/p38 MAPK/NF-κB cascade.

Although numerous studies have demonstrated that titanium dioxide nanoparticles (TiO2 NPs) can be accumulated in various animal organs and can cause toxicity, there is currently only limited data regarding reproductive toxicity especially on the toxic mechanisms of TiO2 NPs in Sertoli cells. In order to investigate the mechanism of reproductive toxicity, primary cultured rat Sertoli cells were exposed to 5, 15, or 30 µg/mL TiO2 NPs for 24 h, and TiO2 NPs internalization, expression of PKC (p-PKC) and p38 MAPK (p-p38 MAPK) as well as calcium homeostasis were examined. Our findings demonstrated that TiO2 NPs crossed the membrane into the cytoplasm or nucleus, and significantly suppressed cell viability of primary cultured rat Sertoli cells in a concentration-dependent manner. Furthermore, immunological dysfunction caused by TiO2 NPs was involved in the increased expression of NF-κB, TNF-α, and IL-1ß, and decreased IκB expression. TiO2 NPs significantly decreased Ca2+ -ATPase and Ca2+ /Mg2+ -ATPase activity and enhanced intracellular Ca2+ levels, and up-regulated the expression of p-PKC and p-p38 MAPK in a dose-dependent manner in primary cultured rat Sertoli cells. Taken together, these findings indicate that TiO2 NPs may induce immunological dysfunction of primary cultured rat Sertoli cells by stimulating the Ca2+ /PKC/p38 MAPK cascade, which triggers NF-κB activation and ultimately induces the expression of inflammatory cytokines in primary cultured rat Sertoli cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1374-1382, 2017.

Nanoparticulate titanium dioxide-inhibited dendritic development is involved in apoptosis and autophagy of hippocampal neurons in offspring mice.

Background: Numerous studies have demonstrated that, upon maternal exposure, nano-TiO2 can cross the placental barrier, accumulate in offspring animals, and cause neurotoxicity. However, the neurotoxic mechanisms are not fully understood. The aim of this study is to determine the effects of nano-TiO2 on the dendritic outgrowth of hippocampal neurons and confirm the role of apoptosis and excessive autophagy in the neurotoxicity of offspring mice caused by nano-TiO2, as well as its molecular mechanisms. Methods: Pregnant mice were intragastrically administered 1, 2, or 3 mg per kg body weight nano-TiO2 consecutively from prenatal day 7 to postpartum day 21. The ultrastructure, mitochondrial membrane potential (MMP), levels of reactive oxygen species (ROS) and peroxides, and ATP contents, along with the expression of apoptosis- and autophagy-related factors, were investigated. Results: The dendritic length of hippocampal neurons was lower in the group treated with nano-TiO2 than in the control group. Apoptosis, excessive autophagy, and nano-TiO2 aggregation in hippocampal neurons resulted from maternal exposure to nano-TiO2. Maternal exposure to nano-TiO2 also resulted in the over-production of ROS, increases in malondialdehyde and protein carbonylation, reductions in MMP and ATP contents, up-regulation of apoptosis- or autophagy-related factors including histone H2AX at serine 139 (γH2AX), cytochrome C (Cyt C), caspase 3, phosphoinositide 3-kinase (PI3K3C), Beclin 1, c-Jun, LC3I, LC3II, JNK and p-JNK expression, and an increase of LC3II/LC3I, as well as down-regulation of Bcl-2 expression in hippocampal neurons of offspring mice. Conclusions: Maternal exposure to nano-TiO2 inhibited the dendritic outgrowth of hippocampal neurons. This effect is closely associated with excessive autophagy, which is related to severe oxidative stress and alterations in the expressions of apoptosis- and autophagy-related factors in the hippocampal neurons of offspring mice, due to maternal exposure to nano-TiO2.

TiO2 nanoparticle-induced neurotoxicity may be involved in dysfunction of glutamate metabolism and its receptor expression in mice.

Titanium dioxide nanoparticles (TiO2 NPs) have been used in environmental management, food, medicine, and industry. But TiO2 NPs have been demonstrated to cross the blood-brain barrier and store up in the brain organization, leading to glutamate-mediated neurotoxicity. However, the neurotoxicity in the brain is not well understood. In this study, mice were exposed to 1.25, 2.5, or 5 mg/kg body weight TiO2 NPs for 9 months, and the glutamate-glutamine cyclic pathway and expressions of glutamate receptors associated with the hippocampal neurotoxicity were investigated. Our findings showed elevations of glutamate release and phosphate-activated glutaminase activity, and reductions in glutamine and glutamine synthetase in the hippocampus following exposure to TiO2 NPs. Furthermore, TiO2 NPs significantly inhibited the expression of N-methyl-d-aspartate receptor subunits (including NR1, NR2A, and NR2B) and metabotropic glutamate receptor 2 in mouse hippocampus. These findings suggest that the imbalance of glutamate metabolism triggered inhibitions of glutamate receptor expression in the TiO2 NP-exposed hippocampus. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 655-662, 2016.

TiO2 nanoparticles-induced apoptosis of primary cultured Sertoli cells of mice.

Titanium dioxide nanoparticles (TiO2 NPs), as largest production and use of nanomaterials, have been demonstrated to have a potential toxicity on reproductive system. However, the mechanism underlying male reproductive toxicity of TiO2 NPs remains limited. Thus, our study was designed to examine the cellular viability, apoptosis, oxidative stress, antioxidant capacity, and expression of apoptotic cytokines in primary cultured Sertoli cells isolated from mice under TiO2 NPs exposure. Results showed that TiO2 NPs exposure from 5 to 30 µg/mL resulted in reduction of cell viability, lactate dehydrogenase release, and induction of apoptosis or death on Sertoli cells. TiO2 NPs could migrate to Sertoli cells, which induced mitochondria-mediated or endoplasmic-reticulum-mediated apoptotic changes including elevation in reactive oxygen species (ROS) generation and reductions in superoxide dismutase, catalase, and glutathione peroxidase activities, decreases in mitochondrial membrane potential (ΔΨm), and releases of cytochrome c into the cytosol. In addition, upregulation of cytochrome c, Bax, caspase-3, glucose-regulated protein 78, and C/EBP homologous protein and caspase-12 protein expression, and downregulation of bcl-2 protein expression in primary cultured Sertoli cells induced by TiO2 NPs treatment. All of the results suggested that ROS generation may play a critical role in the initiation of TiO2 NPs-induced apoptosis by mediation of the disruption of ΔΨm, the cytochrome c release, and further the activation of caspase cascade and unfolded protein response signaling pathway.

Decreased spermatogenesis led to alterations of testis-specific gene expression in male mice following nano-TiO2 exposure.

Although TiO2 nanoparticles (NPs) exposure has been demonstrated to cross blood-testis barrier and accumulate in the testis resulting in the reduction of sperm numbers, limited data with respect to the molecular mechanism of decreased spermatogenesis caused by TiO2 NP exposure. In this research, testicular damage, sperm number and alterations in testis-specific gene expressions in male mice induced by intragastric administration with TiO2 NPs for six months were investigated. It was found out that TiO2 NPs could migrate to cells, deposit in the testis and epididymis and thus cause damages to relevant organs, which are, to be more specific, the reductions of total sperm concentrations and sperm motility and an enhancement in the number of abnormal sperms in the cauda epididymis. Furthermore, the individual expression regarding to the mRNAs and proteins of testis-specific genes, including Cdc2, Cyclin B1, Dmcl, TERT, Tesmin, TESP-1, XPD and XRCCI, were significantly declined, whereas Gsk3-ß and PGAM4 expressions were greatly elevated in mouse testis due to the exposures, which in fact implied that the reduced spermatogenesis may be involved in the alternated testis-specific gene expressions in those exposed male mice.

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2.

Numerous studies have indicated that nano-titanium dioxide (TiO2) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO2 affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 µg/mL nano-TiO2 for 24 h, and nano-TiO2 internalization, dendritic growth, glutamate metabolism, expression of N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (INa) and potassium current (IK) were examined. Our findings demonstrated that nano-TiO2 crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO2 induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca(2+)]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the INa and IK. In addition, nano-TiO2 increased nitric oxide and nitrice synthase, attenuated the activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO2 inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function.