2-activated NF-κB signalling pathway in mice following exposure to titanium dioxide nanoparticles

Journal of Hazardous Materials 313 (2016) 68–77

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Cardiac inflammation involving in PKC␧ or ERK1/2 -activated NF-␬B signalling pathway in mice following exposure to titanium dioxide nanoparticles Xiaohong Yu a , Fashui Hong b,c,∗∗ , Yu-Qing Zhang a,∗ a Department of Applied Biology, School of Basic Medical and Biological Sciences, Soochow University, RM 702-2303, Renai Road No. 199, Dushuhu Higher Edu. Town, Suzhou 215123, China b Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, Huaiyin Normal University, Huaian 223300, China c Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake, Huaiyin Normal University, Huaian 223300, China

h i g h l i g h t s

g r a p h i c a l

• Exposure to TiO2 NPs induced car-

Exposure to TiO2 NPs induces cardiac damage, especially inflammatory response, which is involved in PKC␧ or ERK1/2 -mediated activation of the NF-␬B signalling pathway in mice.

diac damage especially inflammation in mice. • Exposure to TiO2 NPs caused changes of cardiac ATP enzymatic activities and NCX-1 expression in mice. • Exposure to TiO2 NPs induced cardiac CAMK II and ␣1/␤1-AR proteins expression altered in mice. • Exposure to TiO2 NPs resulted in activation of PKC␧ and ERK1/2 medicated NF-␬B signalling pathways in mice.

a r t i c l e

i n f o

Article history: Received 18 November 2015 Received in revised form 15 March 2016 Accepted 30 March 2016 Available online 31 March 2016 Keywords: Titanium dioxide nanoparticles Mice Cardiac damage

a b s t r a c t

a b s t r a c t The evaluation of toxicological effects of nanoparticles (NPs) is increasingly important due to their growing occupational use and presence as compounds in consumer products. Recent researches have demonstrated that long-term exposure to air particulate matter can induce cardiovascular events, but whether cardiovascular disease, such as cardiac damage, is induced by NP exposure and its toxic mechanisms is rarely evaluated. In the present study, when mice were continuously exposed to TiO2 NPs at 2.5, 5 or 10 mg/kg BW by intragastric administration for 90 days, obvious histopathological changes, and great alterations of NF-␬B and its inhibitor I-␬B, as well as TNF-␣, IL-1␤, IL-6 and IFN-␣ expression were induced. The NPs significantly decreased Ca2+ -ATPase, Ca2+ /Mg2+ -ATPase and Na+ /K+ -ATPase activities and enhanced NCX-1 content. The NPs also considerably increased CAMK II and ␣1/␤1-AR expression

∗ Corresponding author. ∗∗ Corresponding author at: Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, Huaiyin Normal University, Huaian 223300, China. E-mail addresses: hongfsh [email protected] (F. Hong), [email protected] (Y.-Q. Zhang). http://dx.doi.org/10.1016/j.jhazmat.2016.03.088 0304-3894/© 2016 Published by Elsevier B.V.

X. Yu et al. / Journal of Hazardous Materials 313 (2016) 68–77 Inflammatory response Molecular mechanisms

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and up-regulated p-PKC␧ and p-ERK1/2 in a dose-dependent manner in the mouse heart. These data suggest that low-dose and long-term exposure to TiO2 NPs may cause cardiac damage such as cardiac fragmentation or disordered myocardial fibre arrangement, tissue necrosis, myocardial haemorrhage, swelling or cardiomyocyte hypertrophy, and the inflammatory response was potentially mediated by NF-␬B activation via the PKC␧ or ERK1/2 signalling cascades in mice. © 2016 Published by Elsevier B.V.

1. Introduction With the development of nanotechnology, nanomaterials have been manufactured worldwide in large quantities and used in wide fields. Titanium dioxide nanoparticles (TiO2 NPs) are novel and widely used nanomaterials that have various uses in extensive areas including pigments [1], paints [2], medicine [3], sunscreen [4], cosmetics [5], food additives [6] and environmental decontamination systems [7,8] due to their high stability, anticorrosion and photocatalytic properties. As the interest in the benefits of TiO2 NPs has increased, there is also increasing concern over their potential adverse effects on human health and animals [9,10]. More attention should be paid to the bio-safety of NPs. Particulate matter exposure is currently a pressing issue in public health, particularly involving effects on the cardiovascular system. Numerous studies have indicated a special correlation between particulate matter exposure and a number of cardiovascular events in humans [11,12] such as myocardial infarct [13,14], atherosclerosis [15,16] and coronary heart disease [17,18]. In recent years, the toxicological properties of TiO2 NPs have been shown in various animal organ types including lung [19–22], liver [23–27], kidney [28–31], spleen [32–37], brain [38,39], hippocampus [40,41], ovary [42,43] and testis [44,45]. Specifically, exposure to TiO2 NPs resulted in cardiovascular disease such as heart damage. For example, heart dysfunction and injury coupled with elevated serum cardiac troponin I and creatine kinase-MB levels in rats under oxidative stress conditions [46], activation of the complement cascade and inflammatory response in C57BL/6 mouse hearts [47] in vivo, and changes in membrane potential, reactive oxygen species generation and cell dysfunction in rat cardiomyocytes in vitro [48,49] were induced by TiO2 NP exposure. Our previous research also suggested that the NPs accumulated in the heart, thus leading to cardiac oxidative damage, inflammation, atherosclerosis, and biochemical dysfunction in mice [50–53] However, TiO2 NP-induced toxicity and its mechanism in heart have yet to be understood deeply. NPs can reportedly cause inflammatory responses that are involved in the activation of some inflammatory cytokines and typical signalling pathway proteins [52,54,55], but whether the TiO2 NP-mediated cardiac inflammation is involved in nuclear factor-␬B (NF-␬B) activation via protein kinase C ␧ (PKC␧) or extracellular signal-regulated kinase (ERK1/2 ) signalling cascades is unclear. PKC␧ is capable of activating several intracellular pathways such as cyclic AMP response element binding protein, nuclear factor (erythroid-derived 2)like 2 and cyclo-oxygenase-2, it can particularly activate NF-␬B, which induces inflammatory responses [20,40,54,56–58]. Moreover, a pro-inflammatory effect of ERK1/2 via NF-␬B activation was observed [55,59–62]. Therefore, we hypothesized that TiO2 NPs may lead to cardiac inflammation that is mediated by PKC␧ or ERK1/2 -activated NF-␬B signalling pathways. The present study was designed to investigate the toxic effect of TiO2 NPs on the heart, especially the induction of the cardiac inflammatory response in mice after continual NP exposure at low doses of 2.5, 5 or 10 mg/kg body weight by intragastric administration for 90 days. To investigate the possible relationship between

TiO2 NP exposure-induced PKC␧ or ERK1/2 -stimulated NF-␬B signalling pathway activation and cardiac inflammation (molecular mechanisms of TiO2 NP toxicity), expression of inflammatory mediators, including NF-␬B, inhibitor of NF-␬B (I-␬B), tumour necrosis factor-␣ (TNF-␣), interleukin-6 (IL-6), interleukin-1␤ (IL-1␤) and interferon-␣ (IFN-␣); activities of Ca2+ -ATPase, Na+ /K+ -ATPase and Ca2+ /Mg2+ -ATPase; and expression of Na+ /Ca2+ exchanger-1 (NCX1), calcium/calmodulin-dependent protein kinase II (CAMK II), ␣1-adrenergic receptor (␣1-AR), ␤1-adrenergic receptor (␤1-AR), p-PKC␧ and p-ERK1/2 proteins, were measured in the mouse heart. 2. Materials and methods 2.1. Chemicals, preparation and characterization Anatase TiO2 NPs were prepared via controlled titanium tetrabutoxide hydrolysis. Details of synthesis and characteristics of TiO2 NPs have been described in our previous work [63]. TiO2 NPs exhibited a peak anatase of 101 by X-ray diffraction. The average particle size of nanoparticulate TiO2 powder suspended in 0.5% w/v hydroxypropylmethylcellulose (HPMC) K4 M solvent after a 24 h (5 mg/mL) incubation ranged from 5 to 6 nm, the surface area was 174.8 m2 /g, the mean hydrodynamic diameter was between 208 nm and 330 nm (mainly 294 nm), and the ␰ potential was 9.28 mV. 2.2. Animals and treatment In total, 80CD-1 (ICR) female mice (20 ± 2 g body weight (BW)) were purchased from the Animal Centre of Soochow University (Suzhou, China). All of the mice were housed in stainless steel cages in a ventilated animal room. The housing facility temperature was maintained at 24 ± 2 ◦ C with a relative humidity of 60 ± 10% and a 12-h light/dark cycle. Distilled water and sterilized food were available for mice ad libitum. Prior to dosing, the mice were acclimated to this environment for 5 days. All of the procedures used in animal experiments conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals [64]. Studies were approved by the Soochow University Institutional Animal Care and Use Committee (Grant 2111270). HPMC (0.5%) was used as a suspending agent. Powdered TiO2 NPs were dispersed onto the HPMC surface, and the suspending solutions containing NPs were ultrasonic treated for 30 min and mechanically vibrated for 5 min before they were used on the mice. The animals were randomly divided into four groups (N = 20): the control group (treated with 0.5% w/v HPMC) and three experimental groups (2.5, 5 and 10 mg/kg BW TiO2 NPs suspensions) and were treated by intragastric administration daily for 90 days. Any symptom or mortality was observed and recorded carefully every day during the 90 days. After 90 days, all of the mice were first weighed and then sacrificed after being ether anaesthesia. Blood samples were collected from the eye vein by removing the eyeball quickly. Serum was harvested by centrifuging blood at 2500 rpm for 10 min. The hearts were excised, weighed and cryopreserved in an ultralow temperature freezer (–80 ◦ C).

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Fig. 1. Histopathological changes in mouse hearts caused by intragastric administration of TiO2 NPs for 90 days (n = 5, average level of 5 mice selected randomly from each group; 400×). (a) Control group (unexposed mice), (b) 2.5 mg/kg TiO2 NPs, (c) 5 mg/kg TiO2 NPs, (d) 10 mg/kg TiO2 NPs. Black arrows suggest fragmentation, interstitial space or disordered myocardial fibre arrangement, blue arrows represent severe fatty degeneration or tissue necrosis, green arrows demonstrate cardiomyocyte swelling or hypertrophy, red arrows denote myocardial haemorrhage or hyperaemia, and yellow circles indicate inflammatory cell infiltration in the myocardial interstitium. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Table 1 Body and cardiac weights and heart coefficients in mice hearts after intragastric TiO2 NPs administration for 90 days. TiO2 NPs (mg/kg BW)

Net increase of BW (g)

Cardiac weight (g)

Heart indices (mg/g)

0 2.5 5 10

24.834 ± 3.532 20.394 ± 2.426** 19.956 ± 2.631** 17.646 ± 3.174***

0.254 ± 0.036 0.233 ± 0.031 0.244 ± 0.043 0.255 ± 0.053

5.844 ± 0.938 5.862 ± 0.796 6.136 ± 0.951 6.898 ± 1.484**

** ***

p < 0.01. p < 0.001. Values represent the mean ± SD (n = 20).

2.3. Body weights and cardiac coefficients

2.5. Cardiac western blots

After weighing the body and hearts, the coefficients of hearts to body weight were calculated as the ratio of heart (wet weight, mg) to body weight (g).

The collected cardiac tissues were lysed in RIPA buffer (Cell Signaling Technology, Inc., USA) containing protease and phosphatase inhibitors (Beijing Solarbio Science & Technology Co., Ltd.) with the help of a bead mill homogenizer. The cardiac homogenate supernatant was separated and collected for protein analysis. Protein concentrations were determined using a standard BCA protein assay kit (Thermo Fisher Scientific, Inc., USA). An equal amount of protein (40 ␮g) for each sample was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and electrophoretically transferred to nitrocellulose (NC) membranes. The membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.05% Tween-20 (PBST) at room temperature for 2 h and then incubated overnight at 4 ◦ C with primary antibodies against NF-␬B (1:1000), anti-I-␬B (1:1000), anti-TNF-␣ (2 ␮g/ml), anti-IL-6 (1:1000), anti-IL-1␤ (0.2 ␮g/ml),

2.4. Histopathological examination of heart For pathologic studies, all of the histopathologic examinations were performed using standard laboratory procedures. The hearts were embedded in paraffin blocks, sliced to 5 ␮m thickness and placed onto glass slides. After haematoxylin–eosin (HE) staining, the stained sections were evaluated using an optical microscope by a histopathologist who had been blinded to the treatments (Nikon U-III Multi-point Sensor System, Japan).

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Fig. 2. Inflammatory cytokine release in mice hearts after intragastric TiO2 NPs administration for 90 days. (A) Representative western blots of TNF-␣, IL-6, IL-1␤, NF-␬B, I-␬B and IFN-␣ in control and TiO2 NP-treated hearts. (B) TNF-␣, IL-6, IL-1␤, NF-␬B, I-␬B and IFN-␣ expression levels in control and TiO2 NP-treated hearts. * p < 0.05, ** p < 0.01 and *** p < 0.001. Values represent the mean ± SD (n = 5, average level of 5 mice selected randomly from each group).

2.7. ELISA for heart protein assessment The supernatants from 10% cardiac tissue homogenates were separated and collected for protein analysis. CAMK II, NCX-1, ␣1-AR and ␤1-AR expression levels were measured using commercial enzyme-linked immunosorbent assay kits (ShangHai HuShang Biological Technology Co., Ltd., P. R. China) according to the manufacturer’s instructions. 3. Statistical analysis Statistical analyses were performed using SPSS 19 software (SPSS, Inc., Chicago, IL, USA). The data were expressed as the means ± SD. One-way analysis of variance was performed to compare the differences of means among multi-group data. Dunnett’s test was performed when each group of experimental data were compared with solvent-control data. Statistical significance for all tests was judged at a probability level of 0.05 (p < 0.05). Fig. 3. Effect of TiO2 NPs on ATP enzyme activities in the mouse heart after intragastric administration of TiO2 NPs for 90 days. * p < 0.05, ** p < 0.01 and *** p < 0.001. Values represent the mean ± SD (n = 5, average level of 5 mice selected randomly from each group).

anti-IFN-␣ (1:400), anti-t/p-PKC␧ (1:500), anti-t/p-ERK1/2 (1:1000) and anti-␤ actin (1:2000) that were obtained from Abcam Trading Co., Ltd. (Shanghai, P. R. China). After washing with PBST three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) for 2 h at room temperature followed by detection using enhanced chemiluminescence (ECL, Millipore). Immunoreactive bands were visualized using X-ray films. The bands were quantified with ImageJ software and normalized to ␤-actin density. 2.6. Analysis of cardiac ATP enzymatic activities To measure their enzymatic activities, the collected cardiac tissues were lysed in 0.9% saline using a bead mill homogenizer. Heart Ca2+ -ATPase, Na+ /K+ -ATPase and Ca2+ /Mg2+ -ATPase activities were assayed with commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). Protein concentrations were determined according to the BCA method.

4. Result 4.1. Body weights and heart coefficients The net increase of BW, cardiac weights and heart coefficients in mouse hearts following exposure to TiO2 NPs for 90 days was exhibited in Table 1. With increasing NP concentration, the net increase of BW significantly decreased, whereas the heart coefficients obviously increased only at the highest dose and there were no alterations in cardiac weights (p < 0.01). Increasing titanium accumulation in the heart was reported previously [50,53] and can potentially contribute to the changes in BW and the cardiac indices. 4.2. Histopathological evaluation Fig. 1 demonstrated the histological photomicrographs of the heart sections caused by TiO2 NP exposure (400×). The TiO2 NP-treated groups exhibited clear cardiac damage such as fragmentation or disordered myocardial fibre arrangement, tissue necrosis and myocardial haemorrhage [Fig. 1(b)–(d)] compared with the control [Fig. 1(a)], while inflammatory cell infiltration in the myocardial interstitium and cardiomyocyte swelling or hypertrophy was only observed in the 5 or 10 mg/kg NP-exposed groups [Fig. 1(c)–(d)]. These results suggested that the histopathological

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Fig. 4. Increased p-PKC␧ and p-ERK1/2 expression in mouse hearts after intragastric TiO2 NP administration for 90 days. *p < 0.05, **p < 0.01 and ***p < 0.001. Values represent the mean ± SD (n = 5, average level of 5 mice selected randomly from each group). Table 2 Increases in CAMK II, NCX-1, ␣1-AR and ␤1-AR expression in mouse hearts after intragastric administration of TiO2 NPs for 90 days. Protein expression (ng/g prot) CAMK II NCX-1 ␣1-AR ␤1-AR * ** ***

TiO2 NPs (mg/kg BW) 0 432.56 ± 57.33 246.68 ± 26.33 486.18 ± 104.60 246.51 ± 60.91

2.5

5

521.14 ± 87.63 269.07 ± 23.78* 652.87 ± 87.27** 264.87 ± 19.95* **

10

542.45 ± 118.83 271.55 ± 17.40* 840.90 ± 75.69*** 301.56 ± 45.79**

**

897.90 ± 26.61*** 323.32 ± 21.02** 1065.75 ± 76.68*** 332.19 ± 8.31**

p < 0.05. p < 0.01. p < 0.001. Values represent the mean ± SD (n = 5, average level of 5 mice selected randomly from each group).

changes of the heart were more significant when the TiO2 NP exposure increased.

increasing doses of TiO2 NPs, CAMK II, NCX-1, ␣1-AR and ␤1-AR levels were increased by 17.00–51.83% (p < 0.05 or 0.01), respectively, compared with the control.

4.3. Inflammatory cytokine expression 4.6. Levels of t/p-PKC␧ and t/p-ERK1/2 proteins The effects of TiO2 NPs on TNF-␣, IL-6, IL-1␤, NF-␬B, I-␬B and IFN-␣ protein expression in the mouse heart are presented in Fig. 2. With increasing NP concentration, TNF-␣, IL-6, IL-1␤, NF-␬B and IFN-␣ expression was significantly elevated by 1.06-1.81-fold, and I-␬B expression was reduced by 1.21–1.55-fold (p < 0.05 or 0.01) compared with the control. 4.4. ATP enzymatic activities Fig. 3 revealed that Ca2+ -ATPase, Na+ /K+ -ATPase and Ca2+ /Mg2+ ATPase activities in the hearts were significantly inhibited by TiO2 NPs with reductions of 5.04%, 7.51% and 32.87%, respectively, for Ca2+ -ATPase; 14.84%, 22.80% and 31.24%, respectively, for Na+ /K+ -ATPase, and 14.63%, 17.68% and 30.46%, respectively, for Ca2+ /Mg2+ -ATPase (p < 0.05, 0.01 or 0.001) compared with the controls. 4.5. PI3K, CAMK2ˇ, NCX-1, ADRA1A and ADRˇ1 expression Table 2 exhibited changes in CAMK II, NCX-1, ␣1-AR and ␤1AR protein expression in the heart after TiO2 NP exposure. With

Elevated protein expression levels of p-PKC␧ and p-ERK1/2 in the mouse hearts after TiO2 NPs exposure is demonstrated in Fig. 4. With increasing NP concentrations, p-PKC␧ activation was increased by 1.29-, 2.09- and 2.34-fold, respectively, p-ERK1 was increased by 1.18-, 1.32- and 1.50-fold, respectively, and p-ERK 2 was increased by 1.40-, 1.65- and 1.70-fold (p < 0.05), respectively, compared with the control. 5. Discussion In the current study, toxicological impacts of TiO2 NPs on mouse hearts were investigated. After intragastric administration of 2.5, 5 and 10 mg/kg BW TiO2 NPs for 90 days, significant reductions in body weight, increased heart indices (Table 1), cardiac damage including fragmentation, interstitial space or disordered myocardial fibre arrangement, fatty degeneration or tissue necrosis, cardiomyocyte swelling or hypertrophy, myocardial haemorrhage or hyperemia, and inflammatory response in the mice (Fig. 1) were observed. Furthermore, we assessed the mechanism of TiO2 NP–induced cardiotoxicity, which is not fully understood.

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Fig. 5. A schematic demonstrating the possible mechanisms of TiO2 NP-induced cardiac damage in mice. ↑: increase or activation, ↓: decrease, and dotted lines arrow: uncertainty.

It is generally recognized that inflammatory cytokines are responsible for the inflammatory response in animals. Our previous studies have demonstrated that the inflammatory response was induced by TiO2 NPs exposure in mouse lungs [20], liver [54], kidney [65], hippocampus [40] and ovary [42], which involved considerable activation of pro-inflammatory mediator TNF-␣, IL-6, IL-1␤ and NF-␬B expression and a significant decrease in I-␬B (NF␬B inhibitor) levels, which is supported by other research [66–68]. NF-␬B is a well-known central nuclear transcription factor that regulates the expression of many genes that are critical for the regulation of inflammation, apoptosis, viral replication, tumorigenesis and various autoimmune diseases. Activation of this transcription factor could be regulated by the phosphorylation of the p65 subunit and degradation of its inhibitor I␬B via IKK␣/␤ phosphorylation, thus resulting its translocation into the nucleus [69]. Activated NF␬B could promote other inflammatory cytokine expression such as TNF-␣, IL-1␤ and IL-6 [65,70]. Type one interferon, IFN-␣ and IFN-␤ (IFN-I) possess antiviral, immunoregulatory and antiproliferative properties. It’s found that IFN-␣ had important role in inflammatory process, and could be stimulated by cellular factors [71]. Niessen et al. reported that elevated IFN-␣ concentrations combined with apoptoticcell-derived membrane microparticles enhanced the secretion of pro-inflammatory cytokines like TNF-␣ and IL-6 by monocytes significantly, whereas absence of IFN-␣ alone did not cause a relevant increase in such cytokines secretion [72]. These results suggest that IFN-␣ may contribute to the initiation and maintenance of inflammation. Cardiac inflammation was observed after TiO2 NPs treatment in mice [47,50,52], but examination of the correlation between the inflammatory mediator expression and TiO2 NP-induced cardiac inflammatory responses have rarely been conducted. In this study, TNF-␣, IL-6, IL-1␤, NF-␬B, I-␬B and IFN-␣ levels were altered in mouse hearts after treatment with different doses of TiO2 NPs in a dose-dependent manner (Fig. 2), which

was in accordance with other reports, implying that the release of pro-inflammatory factors also plays a vital role in progression of the cardiac inflammatory response [Fig. 1(c) and (d)] in mice. However, the activated impact of NF-␬B on IFN-␣ and the activation of inflammatory cytokine expression such as TNF-␣, IL-1␤ and IL-6 by IFN-␣ in the damaged mouse heart due to TiO2 NPs exposure remain uncertain, it will be studied using NF-␬B-/- or IFN-␣-/-mice model in the future. However, what contributes to the altered expression of these inflammatory mediators? Intracellular ion homeostasis, which is related to some ion channels and transporters, is essential for the physiological function of cells. In this article, Ca2+ -ATPase, Na+ /K+ -ATPase and Ca2+ /Mg2+ -ATPase activities in the mouse heart revealed a significant dose-dependent inhibition that was triggered by TiO2 NPs exposure (Fig. 3). The Ca2+ -ATPase was an important Ca2+ binding protein and has considerable capacity in regulating intracellular Ca2+ ([Ca2+ ]i) homeostasis by exporting excessive Ca2+ to extracellular spaces[73]. The Ca2+ /Mg2+ -ATPase, which is concerned with [Ca2+ ]i concentrations, could be activated by millimolar concentrations of Ca2+ or Mg2+ [74]. The Na+ /K+ -ATPase (NKA) is another special ion pump that mediates Na+ efflux and K+ influx and has a fundamental impact to regulate the transmembrane ionic balance, membrane potential, pH balance and cell volume [75]. Particularly in cardiomyocytes, it maintained plasma membrane concentration gradients of K+ , the most crucial determinant of cellular membrane potential, and Na+ , which is the driving force behind many significant ion-exchange processes associated with transmembrane ion transport [76]. The heart is multiply influenced by even small alterations in the intracellular Na+ ([Na+ ]i) content, which regulated by NKA via altering [Ca2+ ]i and pH levels, suggesting that the NKA may play a role in maintaining cardiomyocyte [Ca2+ ]i homeostasis [77]. The Na+ /K+ -ATPase also affected [Ca2+ ]i levels by interacting with the Na+ /Ca2+ exchanger (NCX) [78].

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The NCX-1 that the only NCX isoform presents in the heart strongly influences myocardial contractibility. In the heart, [Na+ ]i are key factors for regulation of Ca2+ cycling, contractility, and cardiac function regulated by Na+ transporters like NCX-1 and Na+ channels. It is reported that the expression of NCX-1 was enhanced in the failing human myocardium [79]. The increased level of [Na+ ]i in rabbit ventricular myocytes with heart failure potentially induced by NCX-1 was found [80]. The relation between elevated NCX-1 expression in dogs heart with chronic mitral valvular insufficiency was indicated [81]. Moreover, some cardiac diseases coupled with enhanced NCX-1 expression were reported [82,83]. These results indicate that NCX-1 may be a cardiac biomarker for monitoring heart risk. NCX-1 is a main Na+ influx and Ca2+ efflux mechanism in cardiomyocytes. However, research from Flesch et al. demonstrated that NCX-1 could also serve in a reverse mode as a source of Ca2+ influx into the cell [79], and the opposite effect was possibly increased by the rise in [Na+ ]I [84,85]. The elevated NCX-1 levels in the mouse hearts after TiO2 NPs exposure was demonstrated in the present paper (Table 2), providing strong evidence that cardiac damage was induced by TiO2 NPs. In addition, it potentially contributed to the increase of [Ca2+ ]i which has rarely been reported in mice heart currently. Therefore, reduced Ca2+ -ATPase, Ca2+ /Mg2+ ATPase and NKA activities and elevated NCX-1 expression in the hearts of mice that have been exposed with TiO2 NPs may alter intracellular ion homeostasis and increase of [Ca2+ ]i concentrations to eventually induce cardiac injury (Fig. 1). Ca2+ , a crucial intracellular second messenger, promoted the various intracellular signal transduction processes in cardiomyocytes [86,87]. In this work, decreased cardiac Ca2+ -ATPase, Ca2+ /Mg2+ -ATPase, NKA activities (Fig. 3) and increased NCX-1 levels (Table 2) in TiO2 NP-treated mice potentially increased [Ca2+ ]i content and subsequently activated signalling proteins such as CAMK II, ERK1/2 and PKC, thereby contributing to heart damage. CAMK II is a multifunctional serine/threonine protein kinase, the activity of which is dependent on [Ca2+ ]i. There is evidence that this kinase was activated by elevated [Ca2+ ]i and plays an important role in regulating intracellular signal transduction [88,89]. Kim et al. suggested that the PKC activation was negatively affected after knocking down CAMK II protein expression, and similar effects were observed with a pharmacological approach [90,91]. These results indicate that CAMK II may contribute to PKC activation. Activation of the mitogen-activated protein kinase (MAPK) family member ERK1/2 by CAMK II has also been reported [88,92]. In this study, exposure to TiO2 NPs induced an obvious dose-dependent increase of CAMK II protein levels in mouse hearts (Table 2), suggesting that it may respond to the elevated [Ca2+ ]i that was caused altered ATPase pump and Ca2+ transporter activities to activate the PKC or ERK1/2 signalling pathways in the heart. ␣1-AR and ␤1-AR protein expression was also enhanced in a dose-dependent manner in the mouse hearts following TiO2 NP exposure (Table 2). Adrenergic receptors (ARs), which are members of the G protein-coupled receptor (GRCP) superfamily, are crucial for cardiac function and are associated with cardiovascular disease. ␤1-AR can activate CAMK II via ␤-arrestin and CaMK II recruitment to its complex at the sarcolemma, where cAMP is generated by adenylate cyclase. Subsequently, cAMP activates Epac, which consequently activates CAMK II [93]. ␣1-AR can also cause polyphosphoinositide hydrolysis as catalysed by phospholipase C (PLC) via the Gq protein family in most tissues [94]. Activation of the phosphatidylinositol pathway, which was downstream of ␣1-AR, resulted in [Ca2+ ]i, release which stimulated CaMK II, and PKC combined with diacylglycerol (DAG) subsequently was also demonstrated by former studies [95,96]. Among the five small G protein subfamilies (Ras, Rho/Rac/Cdc42, Arf/Sar1, Rab and Ran) that acted as molecular switches to regulate numerous cellular responses, Ras and Rho have been more extensively studied in the

heart [97]. The main effectors of Ras proteins are ERK1/2 and phosphatidylinositol 3-kinase (PI3K). In cardiomyocytes, GPCRs can be activated by their ligands such as phenylephrine, angiotensin II and endothelin. Activated GPCRs including the ␣1-AR subsequently activate Ras via a PKC-mediated mechanism [98,99]. Altogether, these findings indicate that ␣1-AR stimulation may activate ERK1/2 via PKC activation, which activates CAMK II via elevating [Ca2+ ]i, and that the regulated impact of ␤1-AR on these signalling cascades is probably through CAMK II activation. PKC is generally regarded as an effector of GPCRs that belongs to a multifunctional serine and threonine protein kinase family. It can be divided to three classes including classical or conventional PKCs, novel PKCs and atypical PKCs. There is considerable evidence that a variety of PKC isoforms act as major modulators of the cardiac damage machinery [100,101]. It’s supported that NF-␬B is a downstream target of the PKC ␧ (a novel PKC) signalling pathway [56,57]. A recent study demonstrated that PKC ␧ activation enhanced NF␬B activity but did not induce pro-inflammatory factor expression, which led to an anti-inflammatory result in vascular endothelial cells [102]. However, in the present work, PKC ␧ (p-PKC ␧) phosphorylation was significantly elevated, indicating PKC ␧ activation (Fig. 4), which was accompanied by increased NF-␬B, TNF-␣, IL-6, IL-1␤ and IFN-␣, expression and decreased I-␬B expression (Fig. 2) in TiO2 NP-exposed mouse hearts. MAPKs, which are composed of three main members ERK1/2 , JNK and p38, are associated with the inflammatory response via NF-␬B activation [55]. ERK1/2 phosphorylation activated redox-sensitive transcription factors such as NF-␬B [62]. A significant increase in TiO2 NP-induced ERK1/2 phosphorylation (p-ERK1/2 ) was demonstrated in mouse hearts in this paper (Fig. 4), which was in accordance with these findings. Therefore, TiO2 NP exposure resulted in cardiac inflammation, which may be related to PKC ␧ and ERK1/2 activation via their stimulation of NF␬B. The possible mechanism of TiO2 NP-induced cardiac damage, especially the inflammatory response, is depicted in Fig. 5.

6. Conclusions Taken together, these findings suggest that TiO2 NP exposure induced fragmentation or disordered arrangement of myocardial fibres, tissue necrosis, myocardial haemorrhage, cardiomyocyte swelling or hypertrophy and an inflammatory response in the mouse heart in a dose-dependent manner, which was closely related to increased NF-␬B and pro-inflammatory cytokine expression, including TNF-␣, IL-6, IL-1␤ and IFN-␣ combined with decreased I-␬B levels. Furthermore, the TiO2 NP-mediated cardiac toxicity was associated with the reduced Ca2+ -ATPase, Na+ /K+ ATPase and Ca2+ /Mg2+ -ATPase activities and elevated NCX-1 concentrations as well as activation of CAMK II, ␣1-AR, ␤1-AR, PKC ␧ and ERK1/2 . Therefore, our findings imply that cardiac lesions induced by TiO2 NP exposure may be mediated by NF-␬B activation via the PKC␧ or ERK1/2 signalling cascades in mice, which will provide a deep understanding of TiO2 NP-induced effects on the cardiovascular system, especially on the heart and its potential action mechanisms, which will arouse attention for nanomaterial applications.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81473007, 81273036, 30901218). The authors gratefully acknowledge the earmarked fund (CARS-22ZJ0504) from the China Agriculture Research System (CARS) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China.

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