Mechanisms of titanium dioxide nanoparticle‐induced oxidative stress and modulation of plasma glucose in mice

Titanium dioxide nanoparticles (TiO₂ NPs) are reported to increase plasma glucose levels in mice at specific doses. The production and accumulation of reactive oxygen species (ROS) is potentially the most important factor underlying the biological toxicity of TiO₂ NPs but the underlying mechanisms are unclear at present. Data from genome‐wide analyses showed that TiO₂ NPs induce endoplasmic reticulum (ER) stress and ROS generation, leading to the inference that TiO₂ NP‐induced ER stress contributes to enhancement of ROS in mice. Resveratrol (Res) effectively relieved TiO₂ NP‐induced ER stress and ROS generation by ameliorating expression of a common set of activated genes for both processes, signifying that ER stress and ROS are closely related. TiO₂ NP‐induced ER stress occurred earlier than ROS generation. Upon treatment with 4‐phenylbutyric acid to relieve ER stress, plasma glucose levels tended toward normal and TiO₂ NP increased ROS production was inhibited. These results suggest that TiO2 NP‐induced ER stress promotes the generation of ROS, in turn, triggering increased plasma glucose levels in mice. In addition, Res that displays the ability to reduce ER stress presents a dietary polyphenol antioxidant that can effectively prevent the toxicological effects of TiO₂ NPs on plasma glucose metabolism.     

The toxic effects of titanium dioxide nanoparticles on plasma glucose metabolism are more severe in developing mice than in adult mice

Titanium dioxide nanoparticles (TiO₂ NPs) are authorized food additives, and children have the highest exposure. Therefore, children are likely more susceptible to the adverse effects of TiO₂ NPs than adults. Previous study showed that oral administration of 50 mg/kg body weight (bw) TiO₂ NPs increase plasma glucose in mice. However, few studies have directly compared the adverse effects of exposure to TiO₂ NPs on plasma glucose metabolism of different age groups. In this study, the developing (age 3 weeks) and adult mice (age 10 weeks) were orally administered with 50 mg/kg bw TiO₂ NPs per day. The TiO₂ NPs induced hyperglycemia earlier in the developing mice than in the adult mice. Then mechanisms were analyzed after mice were oral administration of TiO2 NPs for 8 weeks and 26 weeks, respectively. Results showed that the treatment with TiO₂ NPs activated xenobiotic biodegradation in livers of both developing and adult mice at the early stage. However, only in the developing mice, TiO₂ NPs induced endoplasmic reticulum (ER) stress in livers and increased reactive oxygen species in livers and sera in the early stage. The ER stress and ROS activated an inflammation response and mitogen‐activated protein kinase pathways, thereby inducing insulin resistance in the livers of developing mice at the early stage. The response of the adult mice was delayed, and these changes were observed in the late stage of the study. The results of this study all suggest that children are more susceptible than adults to the toxicity of orally administered TiO₂ NPs.     

RNA sequencing analysis shows that titanium dioxide nanoparticles induce endoplasmic reticulum stress,which has a central role in mediating plasma glucose in mice

Titanium dioxide nanoparticles (TiO₂ NPs) constitute the top five NPs in use today. In this study, oral administration of 50, 100, and 200?mg/kg body weight (b.w.) TiO₂ NPs increases plasma glucose in mice, whereas 10 and 20?mg/kg b.w. TiO₂ NPs did not. RNA sequencing (RNA-seq) technology was used to investigate genome-wide effects of TiO₂ NPs. Clustering analysis of the RNA-seq data showed the most significantly enriched gene ontology terms and KEGG pathways related to the endoplasmic reticulum (ER) and ER stress. Molecular biology verification showed that 50?mg/kg b.w. and higher doses TiO₂ NPs activated a xenobiotic biodegradation response and increased expression of cytochrome P450 family genes in mouse livers, thus inducing ER stress in mice. ER stress-activated MAPK and NF-κB pathways and induced an inflammation response, resulting in phosphorylation of the insulin receptor substrate 1 and, consequently, insulin resistance. This was the main mechanism by which TiO₂ NPs increased plasma glucose in mice. Meanwhile, ER stress disturbed the monooxygenase system, and thus generated reactive oxygen species (ROS). Relief of ER stress with 4-phenylbutyric acid inhibited all the above effects of TiO₂ NPs, including the generation of ROS. Therefore, TiO₂ NP-induced ER stress was a decisive factor with a central role in plasma glucose disturbance in mice.     

Reactive oxygen species‐dependent JNK downregulated olaquindox‐induced autophagy in HepG2 cells

Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as “type II programmed cell death,” and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase‐9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox‐treated HepG2 cells. Olaquindox‐induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy‐related MAP‐LC3 and Beclin 1 proteins. The autophagy inhibitor 3‐methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl‐2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N‐acetyl‐L ‐cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c‐Jun N‐terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox‐induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox‐induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox‐induced apoptosis can be enhanced by 3‐methyladenine. Olaquindox‐induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS‐dependent JNK. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of diltiazem on plasma glucose,insulin and glucagon during an oral glucose tolerance test in healthy volunteers

Summary The effect of the calcium antagonist, diltiazem, on glycoregulation was investigated in 12 healthy volunteers using a standard glucose tolerance test. No significant change was observed in plasma glucose, insulin or glucagon levels after oral treatment for B days with diltiazem 180 mg/day.     

Effects of oral administration of titanium dioxide fine-sized particles on plasma glucose in mice

Titanium dioxide (TiO₂) is an authorized additive used as a food colorant, is composed of nano-sized particles (NP) and fine-sized particles (FP). Previous study reported that oral administration of TiO₂ NPs triggers an increase in plasma glucose of mice. However, no previous studies have focused on toxic effects of TiO₂ FPs on plasma glucose homeostasis following oral administration. In the current study, mice were orally administered TiO₂ FPs greater than 100 nm in size (64 mg/kg body weight per day), and effects on plasma glucose levels examined. Our results showed that titanium levels was not changed in mouse blood, livers and pancreases after mice were orally administered TiO₂ FPs. Biochemical analyzes showed that plasma glucose and ROS levels were not affected by TiO₂ FPs. Histopathological results showed that TiO₂ FPs did not induce pathology changes in organs, especially plasma glucose homeostasis regulation organs, such as pancreas and liver. Western blotting showed that oral administration of TiO₂ FPs did not induce insulin resistance (IR) in mouse liver. These results showed that, TiO₂ FPs cannot be absorbed via oral administration and affect plasma glucose levels in mice.     

The effect and underlying mechanisms of titanium dioxide nanoparticles on glucose homeostasis: A literature review

Titanium dioxide (TiO₂) is used extensively as a white pigment in the food industry, personal care, and a variety of products of everyday use. Although TiO₂ has been categorized as a bioinert material, recent evidence has demonstrated different toxicity profiles of TiO₂ nanoparticles (NPs) and a potential health risk to humans. Studies indicated that titanium dioxide enters the systemic circulation and accumulates in the lungs, liver, kidneys, spleen, heart, and central nervous system and may cause oxidative stress and tissue damage in these vital organs. Recently, some studies have raised concerns about the possible detrimental effects of TiO₂ NPs on glucose homeostasis. However, the findings should be interpreted with caution due to the methodological issues. This article aims to evaluate current evidence regarding the effects of TiO₂ NPs on glucose homeostasis, including possible underlying mechanisms. Furthermore, the limitations of current studies are discussed, which may provide a comprehensive understanding and new perspectives for future studies in this field.     

Silver nanoparticles induced reactive oxygen species via photosynthetic energy transport imbalance in an aquatic plant

The rapid growth in silver nanoparticles (AgNPs) commercialization has increased environmental exposure, including aquatic ecosystem. It has been reported that the AgNPs have damaging effects on photosynthesis and induce oxidative stress, but the toxic mechanism of AgNPs is still a matter of debate. In the present study, on the model aquatic higher plant Spirodela polyrhiza, we found that AgNPs affect photosynthesis and significantly inhibit Photosystem II (PSII) maximum quantum yield (F_v/F_m) and effective quantum yield (Φ_PSII). The changes of non-photochemical fluorescence quenching (NPQ), light-induced non-photochemical fluorescence quenching [Y(NPQ)] and non-light-induced non-photochemical fluorescence quenching [Y(NO)] showed that AgNPs inhibit the photo-protective capacity of PSII. AgNPs induce reactive oxygen species (ROS) that are mainly produced in the chloroplast. The activity of ribulose-1, 5-bisphosphate carboxylase–oxygenase (Rubisco) was also very sensitive to AgNPs. The internalized Ag, regardless of whether the exposure was Ag^+?or AgNPs had the same capacity to generate ROS. Our results support the hypothesis that intra-cellular AgNP dissociate into high toxic Ag⁺. Rubisco inhibition leads to slowing down of CO₂ assimilation. Consequently, the solar energy consumption decreases and then the excess excitation energy promotes ROS generation in chloroplast.     

Effects of clofibrate on glucose tolerance,serum insulin,serum lipoproteins and plasma fibrinogen

Summary The effects of clofibrate therapy were studied in fifty year old, weight-stable, free-living men not on specific dietary treatment. The patients had been selected because of hyperlipidaemia and/or glucose intolerance found at a health screening survey. The study lasted for four months and was double-blind and cross-over in design. The results showed no effect of clofibrate treatment on glucose tolerance, as measured by an intravenous glucose tolerance test, neither in the entire group (t=0.90, 2p>0.05, n=21) nor in a subgroup (t=–0.71, 2p>0.05, n=10) with glucose intolerance on screening. Fasting blood glucose was significantly decreased (t=3.71, 2p<0.01) in the subgroup (n=8) with fasting blood glucose greater than 100 mg/100 ml during the placebo period. There was, however, a significant (t=4.00, 2p<0.001) reduction in fasting serum(S-)insulin concentration during clofibrate therapy. The reduction was correlated with relative body weight (r=0.59, 2p<0.01). Elevated S-lipoproteins were decreased by clofibrate therapy, S-low density lipoprotein cholesterol concentrations by 20 per cent and S-very low density lipoprotein triglyceride concentrations by 52 per cent. Significant falls in plasma fibrinogen concentration (t=4.57, 2p<0.001) and erythrocyte sedimentation rate (t=2.48, 2p<0.025) also occured, which were not correlated to the other effects reported.     

Effect of chlorpromazine on blood glucose and plasma insulin in man

Summary In three groups of normal subjects and in one group of patients with latent diabetes mellitus a study has been made of the effects of chlorpromazine (CPZ) on blood glucose and plasma insulin. CPZ 75 mg/day for 7 days did not alter the plasma insulin response after oral glucose; nor did CPZ 50 mg/day for 7 days affect the glucose assimilation rate or insulin response to glucose injection. Infusion of CPZ 50 mg in 60 min slightly increased the basal blood glucose level but had no significant effect on basal plasma insulin. The insulin/glucose ratio after the end of the infusion was significantly higher than during the period of infusion of the drug. In latent diabetic patients CPZ infusion significantly diminished the insulin/glucose ratio during an intravenous glucose tolerance test. These results suggest that, whereas prolonged treatment with low doses of CPZ did not modify glucose tolerance and glucose-stimulated pancreatic response, higher acute doses of the drug may induce hyperglycaemia and can inhibit insulin secretion both in normal man and in patients with latent diabetes mellitus.     

高甘油三酯并糖调节异常及正常者的胰岛素抵抗及胰岛素分泌的研究

目的:了解高甘油三酯并糖调节异常(IGR)[包括空腹血糖受损(IFG)或/和糖耐量减低(IGT)者]及正常(NGT)者的胰岛素抵抗及胰岛素分泌。方法:在体检人群中筛选出高甘油三酯并IGR及NGT者62人及健康者14人为研究对象,测血压、身高、体重、腰围、臀围,计算体重指数(BMI)及腰臀比(腰/臀)。经口服葡萄糖耐量试验(OGTT)测血糖(PG),同时检测甘油三酯(TG)及真胰岛素(TINS)、胰岛素原(PINS),根据血糖和TG结果将研究对象分为四组。①组(健康对照组):为NGT和TG正常者14人,②组(NGT+HTG):为NGT而高TG者即20人,③组(IFG/IGT+HTG):IFG或IGT伴高TG者23人,④组(IFG+IGT+HTG):IFG并IGT伴高TG者19人,四组均排除高血压病、肥胖及其它疾病。根据稳态模型公式计算各组的胰岛素抵抗指数(HOMA-IR),HOMA胰岛β细胞功能指数(HOMA-β),胰岛素快速反应指数(AIR)。结果:四组的BMI、腰/臀,真胰岛素,胰岛素原水平呈现①②③④组依次递增的趋势,③④组的HOMA-IR有别于①②组,四组的HOMA-β依次递减,差异均有显著性,P<0.05~0.000。结论:高甘油三酯并IGR及NGT者已出现胰岛素抵抗、胰岛素分泌及胰岛β细胞功能变化。     

Perfluorooctane sulfonate induces apoptosis in lung cancer A549 cells through reactive oxygen species‐mediated mitochondrion‐dependent pathway

Perfluorooctane sulfonate (PFOS) is a widespread environmental contaminant that is detected in the lung of mammals. The mechanisms underlying PFOS‐induced lung cytotoxicity remain unclear. The main purpose of this study was to evaluate the cytotoxic effects of PFOS on human lung cancer A549 cells and its possible molecular mechanism. A549 cells were treated with PFOS (0, 25, 50, 100 and 200 μm ) and the cellular apoptosis, mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In this study, PFOS induced a dose‐dependent increase in A549 cell toxicity via an apoptosis pathway as characterized by increased percentage of sub‐G1, activation of caspase‐3 and ?9, and increased ratio of Bax/bcl‐2 mRNA expression. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N‐Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, can effectively block PFOS‐induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicate that PFOS induces apoptosis in A549 cells through a reactive oxygen species‐mediated mitochondrial dysfunction pathway mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

Tissue distribution and histopathological effects of titanium dioxide nanoparticles after intravenous or subcutaneous injection in mice

Nanoparticles can be formed following degradation of medical devices such as orthopedic implants. To evaluate the safety of titanium alloy orthopedic materials, data are needed on the long‐term distribution and tissue effects of injected titanium nanoparticles in experimental animals. In this study, we evaluated the tissue distribution and histopathological effects of titanium dioxide (TiO₂) nanoparticles (approximately 120 nm diameter) in mice after intravenous (i.v.; 56 or 560 mg kg^?1 per mouse) or subcutaneous (s.c.; 560 or 5600 mg kg^?1 per mouse) injection on two consecutive days. Animals were examined 1 and 3 days, and 2, 4, 12 and 26 weeks after the final injection. When examined by light microscopy, particle agglomerates identified as TiO₂ were observed mainly in the major filtration organs – liver, lung and spleen – following i.v. injection. Particles were still observed 26 weeks after injection, indicating that tissue clearance is limited. In addition, redistribution within the histological micro‐compartments of organs, especially in the spleen, was noted. Following s.c. injection, the largest particle agglomerates were found mainly in the draining inguinal lymph node, and to a lesser extent, the liver, spleen and lung. With the exception of a foreign body response at the site of s.c. injection and the appearance of an increased number of macrophages in the lung and liver, there was no histopathological evidence of tissue damage observed in any tissue at any time point. Published 2011. This article is a US Government work and is in the public domain in the USA.     

Effects of SiO2 nanoparticles on HFL‐I activating ROS‐mediated apoptosis via p53 pathway

Silicon dioxide nanoparticles (SiO₂ NPs) have attracted increasing interest as nanovehicles for delivering drugs, genes and bio‐active molecules into cells. However, it is still unknown whether SiO₂ NPs could cause side‐effects to normal cells. In the present study, human lung fibroblasts (HFL‐Is) were directly exposed to two different sizes of SiO₂ NPs. The effect of size and concentration on cell response was studied by analyzing the cell viability, the ratio of apoptosis and the pathway of cell injury. The results demonstrated that a size‐associated and a dose‐dependent toxicity of HFL‐Is was induced by SiO₂ NPs. Meanwhile, the expression of reactive oxygen species in HFL‐I was significantly increased. This activation effect was accompanied by upregulation of p53 expression, release of cytochrome C from chondriosomes, inhibition of Bcl2, and activation of Bax and caspase 9. These findings implied that SiO₂ NPs might induce apoptosis of HFL‐Is by stimulating reactive oxygen species release and subsequently causing the activation of p53 pathway in vitro. Copyright © 2011 John Wiley & Sons, Ltd.     

Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25-200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion of glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level.     

Metabolic profiling reveals disorder of carbohydrate metabolism in mouse fibroblast cells induced by titanium dioxide nanoparticles

As titanium dioxide (TiO₂) nanoparticles are widely used commercially, their potential biosafety and metabolic mechanism needs to be fully explained. In this study, the cytotoxicity of homogeneous and weakly aggregated (< 100 nm) TiO₂ nanoparticles was investigated by analyzing the changes in metabolite profiles both in mouse fibroblast (L929) cells and their corresponding culture media using gas chromatograph with a time‐of‐flight mass spectrometry (GC/TOFMS)‐based metabolomic strategy. With multivariate statistics analysis, satisfactory separations were observed in principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS‐DA) models. Based on the variable importance in the OPLS‐DA models, a series of differential metabolites were identified by comparison between TiO₂ nanoparticle‐treated L929 cells or their corresponding culture media and the control groups. It was found that the major biochemical metabolism (carbohydrate metabolism) was suppressed in TiO₂ nanoparticle‐treated L929 cells and their corresponding culture media. These results might account for the serious damage to energy metabolism in mitochondria and the increased cellular oxidation stress in TiO₂ nanoparticle‐induced L929 cells. These results also suggest that the metabolomic strategy had a great potential in evaluating the cytotoxicity of TiO₂ nanoparticles and thus was very helpful in understanding its underlying molecular mechanisms. Copyright © 2012 John Wiley & Sons, Ltd.     

氯诺昔康围术期镇痛对患者血糖、胰岛素和皮质醇的影响

目的观察氯诺昔康围术期镇痛对机体血糖、胰岛素和血浆皮质醇水平的影响。方法24例在硬膜外麻醉下行择期胆囊切除术病人,随机分为氯诺昔康组(L组):经硬膜外腔单次推注吗啡1.5 mg和新斯的明1 mg并复合静脉推注氯诺昔康镇痛;对照组(C组):经硬膜外腔单次推注吗啡1.5 mg和新斯的明1 mg镇痛。于术前(T1)、术毕(T2)、术后第1天(T3)、术后第2天(T4)和术后第3天(T5)测定血糖、胰岛素和血浆皮质醇浓度,视觉模拟评分法(VAS)作术后4、24和48 h的静止与活动时VAS评分。结果C组术后4 h、术后24 h静止或活动VAS评分明显高于L组(P<0.05)。C组术毕、术后第1天、术后第2天血浆皮质醇浓度与术前比明显升高(P<0.05或0.01),而L组血浆皮质醇浓度仅术毕时显著增高(P<0.05);L组术毕、术后第1天、术后第2天血糖、血浆胰岛素和血浆皮质醇水平均显著低于C组(P<0.05或0.01)。结论氯诺昔康应用于围术期镇痛,增强硬膜外吗啡的镇痛效果,有效地遏制机体的高代谢状态,减轻术后糖代谢紊乱。     

The effects of ingestion time of gliclazide in relationship to meals on plasma glucose,insulin and c-peptide levels

Summary The effect of altering the timing of gliclazide administration in relation to a meal was studied in ten type 2 (non-insulin dependent) chronically treated diabetics. Gliclazide was given 30 min before, at the start of and 30 min after breakfast or omitted altogether. Plasma gliclazide was present at greater than 2 mg/l throughout the study periods. Administration at 30 min after the meal significantly delayed the time to peak for plasma gliclazide. No significant difference was noted in plasma glucose, insulin or c-peptide patterns with any protocol. It is concluded that, in clinical practice, with chronically treated diabetics the timing of gliclazide ingestion in relation to meals is not critical.     

Thymoquinone suppresses the proliferation of renal cell carcinoma cells via reactive oxygen species‐induced apoptosis and reduces cell stemness

Thymoquinone is a phytochemical compound isolated from Nigella sativa and has various biological effects, including anti‐inflammation, antioxidation, and anticancer. Here, we further investigated the anticancer effects and associated molecular mechanism of 2‐methyl‐5‐isopropyl‐1,4‐benzoquinone (thymoquinone) on human renal carcinoma cell lines 786‐O and 786‐O‐SI3 and transitional carcinoma cell line BFTC‐909. Results showed that thymoquinone significantly reduced cell viability, inhibited the colony formation of renal cancer cells, and induced cell apoptosis and mitochondrial membrane potential change in both cancer cells. In addition, thymoquinone also triggered the production of reactive oxygen species (ROS) and superoxide and the activation of apoptotic and autophagic cascade. ROS inhibition suppressed the caspase‐3 activation and restored the decreased cell viability of 786‐O‐SI3 in response to thymoquinone. Autophagy inhibition did not restore the cell viability of 786‐O‐SI3 suppressed by thymoquinone. Moreover, thymoquinone suppressed the cell sphere formation and the expression of aldehyde dehydrogenase, Nanog, Nestin, CD44, and Oct‐4 in 786‐O‐SI3 cells. The tumor‐bearing model showed that thymoquinone in vivo inhibited the growth of implanted 786‐O‐SI3 cell. All these findings indicate that thymoquinone inhibits the proliferation of 786‐O‐SI3 and BFTC‐909 cell possibly due to the induction of ROS/superoxide and the consequent apoptosis, suggesting that thymoquinone may be a potential anticancer supplement for genitourinary cancer.     

Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles

With large-scale production and wide application of nanoscale ZnO, its health hazard has attracted extensive worldwide attention. In this study, cytotoxicity of different sized and shaped ZnO nanoparticles in mouse macrophage Ana-1 was investigated. And contribution of dissolved Zn(2+) and ROS in toxicity of ZnO particles was analyzed. The results indicated that ZnO particles manifested dose-dependent toxic effect on Ana-1 cells without size-dependence, and the particles shape may impact cytotoxicity of ZnO particles. When the concentration of dissolved Zn(2+) tended to equilibrium in the complete cell medium, the zinc ion concentration was approximately 10 μg/ml, inducing about 50% cell death, which was close to the cytotoxicity of ZnCl(2) (IC(50)=13.33 μg Zn/ml). The Zn(2+) concentration had significant correlations with cell viability and LDH level induced by the supernatant of ZnO particle suspensions (incubation at 37°C for 24h). Thus, the dissolved Zn(2+) played the main role in toxic effect of ZnO particles. Moreover, ROS generation assays demonstrated that ZnO particles produced intrinsically a small quantity of ROS, intracellular ROS was mainly produced after ZnO particles or the dissolved Zn(2+) entered into the cells. Although intracellular ROS had significant correlations with cell viability and LDH induced by ZnO particles, intracellular ROS may not be a major factor in cytotoxicity of ZnO nanoparticles, but the cytotoxic response.