Sulfur amino acid deprivation in cadmium chloride toxicity in hepatoma cells

The aim of this study was to investigate the effect of individual sulfur amino acid deprivation in cadmium chloride toxicity. HTC cells were deprived of cystine and/or methionine for 12 h and then exposed to CdCl₂ for 12 h. HepG2 cells were deprived of cystine for 3 and 5 h and exposed to CdCl₂ for 3 h. In addition HepG2 cells were deprived of methionine for 12 h and then exposed to CdCl₂ for 5 and 12 h. Our results indicate that only cystine depletion increased cadmium toxicity in HTC cells but not in HepG2 cells as indicated by the neutral red assay. This effect was due to glutathione depletion as indicated by measurement of intracellular glutathione in HTC cells following deprivation of cystine. Methionine depletion had only a slight effect on the viability of HepG2 cells.     

Modulation of cadmium chloride toxicity by sulphur amino acids in hepatoma cells.

Cadmium is a toxic metal and no effective antidote exists at present. The aim of this study was to examine whether sulphur amino acids, involved in glutathione synthesis, can modulate cadmium toxicity in vitro. Two hepatoma cell lines (HepG2 and HTC cells) were exposed to cadmium chloride (0-100 microM) for 8h in control media or in media containing 1mM of homocysteine, cysteine or cystathionine. Cell viability was then assessed with the neutral red assay. In order to assess the mechanism by which homocysteine and cysteine modulate cadmium toxicity their ability to scavenge reactive oxygen species was determined as well as the potential to increase intracellular glutathione levels. The ability of the sulphur amino acids to prevent cadmium uptake by HTC and HepG2 cells was also assessed. The results indicate that homocysteine and cysteine protect efficiently both cell lines from cadmium chloride toxicity whereas cystathionine protects efficiently HTC cells but not HepG2 cells. This effect was shown to be dependent on the dose of each amino acid and increased protection from cadmium was observed with increasing concentrations of homocysteine and cysteine. Both amino acids prevented the formation of reactive oxygen species only when they were administered together with cadmium chloride. In addition homocysteine and cysteine did not increase intracellular glutathione levels. The results indicate that the mechanism by which sulphur amino acids protect from cadmium toxicity in vitro is due to the reduced uptake of the metal by the cells possibly by direct binding to the -SH group of the amino acids.     

Comparison of in vitro toxicity of silver ions and silver nanoparticles on human hepatoma cells

Scientific information on the potential harmful effects of silver nanoparticles (AgNPs) on human health severely lags behind their exponentially growing applications in consumer products. In assessing the toxic risk of AgNP usage, liver, as a detoxifying organ, is particularly important. The aim of this study was to explore the toxicity mechanisms of nano and ionic forms of silver on human hepatoblastoma (HepG2) cells. The results showed that silver ions and citrate‐coated AgNPs reduced cell viability in a dose‐dependent manner. The IC50 values of silver ions and citrate‐coated AgNPs were 0.5 and 50 mg L^?1, respectively. The LDH leakage and inhibition of albumin synthesis, along with decreased ALT activity, indicated that treatment with either AgNP or Ag ions resulted in membrane damage and reduced the cell function of human liver cells. Evaluation of oxidative stress markers demonstrating depletion of GSH, increased ROS production, and increased SOD activity, indicated that oxidative stress might contribute to the toxicity effects of nano and ionic forms of silver. The observed toxic effect of AgNP on HepG2 cells was substantially weaker than that caused by ionic silver, while the uptake of nano and ionic forms of silver by HepG2 cells was nearly the same. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 679–692, 2016.     

Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells

Cytotoxicity induced by silver nanoparticles (AgNPs) and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells. Toxicity induced by silver (Ag⁺) ions was studied in parallel using AgNO₃ as the Ag⁺ ion source. Using cation exchange treatment, we confirmed that the AgNP solution contained a negligible amount of free Ag⁺ ions. Metal-responsive metallothionein 1b (MT1b) mRNA expression was not induced in AgNP-treated cells, while it was induced in AgNO₃-treated cells. These results indicate that AgNP-treated cells have limited exposure to Ag⁺ ions, despite the potential release of Ag⁺ ions from AgNPs in cell culture. AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and induced intracellular oxidative stress. AgNPs exhibited cytotoxicity with a potency comparable to that of Ag⁺ ions in in vitro cytotoxicity assays. However, the toxicity of AgNPs was prevented by use of the antioxidant N-acetylcysteine, and AgNP-induced DNA damage was also prevented by N-acetylcysteine. AgNO₃ treatment induced oxidative stress-related glutathione peroxidase 1 (GPx1) and catalase expression to a greater extent than AgNP exposure, but treatment with AgNO₃ and AgNPs induced comparable superoxide dismutase 1 (SOD1) expression levels. Our findings suggest that AgNP cytotoxicity is primarily the result of oxidative stress and is independent of the toxicity of Ag⁺ ions.     

Protective effect of sodium molybdate against the acute toxicity of cadmium chloride

Pretreatment of rats with Na₂MoO₄ (1.24 mmol/kg, once a day for 3 days, i.p.) partially protected them againts the acute toxicity of CdCl₂ (0.075 mmol/kg, once, s.c., 24 h after pretreatment with Na₂MoO₄). The survival number of rats per total number of rats in the CdCl₂-dosed group was 10/10, 8/10, 6/10, 2/10 and 0/10 on 0, 1, 2, 6 and 18 days after treatment with CdCl₂ whereas in the group where CdCl₂ is given after pretreatment with Na₂MoO₄ it is 10/10 and 6/10 on 0 and 18 days. The body weight of CdCl₂-dosed rats consistently decreased until their death while that of Na₂MoO₄- CdCl₂-dosed rats similarly decreased up to 4 days after exposure to CdCl₂ but then increased almost normally. In order to elucidate the mechanism of protective action of Na₂MoO₄ against the acute toxicity of CdCl₂, cellular components such as DNA, inorganic cations and metallothionein were measured in the liver after exposure to CdCl₂. The treatment with CdCl₂ alone reduced K content and increased Ca content but pretreatment with Na₂MoO₄ prevented such alterations in the levels of those cations caused by CdCl₂. Metallothionein content in the liver was significantly elevated in the CdCl₂-treated groups as compared to saline controls although the protein content was higher in the Na₂MoO₄-CdCl₂-dosed group than in the CdCl₂-dosed group. There was no difference in the protein content of the liver between saline controls and the Na₂MoO₄-dosed group. This suggests that Na₂MoO₄ alleviated the acute toxicity of CdCl₂ in the rat and the protective mechanism by the metal is in part related to the enhancement of liver Cd-metallothionen induction.     

Interactive toxicity of usnic acid and lipopolysaccharides in human liver HepG2 cells

Usnic acid (UA), a natural botanical product, is a constituent of some dietary supplements used for weight loss. It has been associated with clinical hepatotoxicity leading to liver failure in humans. The present study was undertaken to evaluate the interactive toxicity, if any, of UA with lipopolysaccarides (LPS), a potential contaminant of food, at low non‐toxic concentrations. The human hepatoblastoma HepG2 cells were treated with the vehicle control and test agents, separately and in a binary mixture, for 24 h at 37 ⁰C in 5% CO₂. After the treatment period, the cells were evaluated by the traditional biochemical endpoints of toxicity in combination with the toxicogenomic endpoints that included cytotoxicity, oxidative stress, mitochondrial injury and changes in pathway‐focused gene expression profiles. Compared with the controls, low non‐toxic concentrations of UA and LPS separately showed no effect on the cells as determined by the biochemical endpoints. However, the simultaneous mixed exposure of the cells to their binary mixture resulted in increased cytotoxicity, oxidative stress and mitochondrial injury. The pathway‐focused gene expression analysis resulted in the altered expression of several genes out of 84 genes examined. Most altered gene expressions induced by the binary mixture of UA and LPS were different from those induced by the individual constituents. The genes affected by the mixture were not modulated by either UA or LPS. The results of the present study suggest that the interactions of low nontoxic concentrations of UA and LPS produce toxicity in HepG2 cells. Published 2012. This article is a US Government work and is in the public domain in the USA.     

Comparison of the sensitivity of different toxicological endpoints in Caco-2 cells after cadmium chloride treatment

The human colorectal adenocarcinoma cell line Caco-2 is a widely used in vitro model of the intestinal barrier. Cadmium chloride (CdCl₂) is a highly toxic metal compound, ubiquitous in the biosphere, able to enter the food chain and to reach the intestinal epithelium, causing structural and functional damages. The aim of this work was to characterise cadmium toxicity in Caco-2 cells and, in particular, to compare the sensitivity of different endpoints revealing damage both on the epithelial barrier and at the cellular or molecular level. After 24-h exposure of the cells to CdCl₂, lactate dehydrogenase (LDH) leakage showed cadmium-induced cell toxicity, significant from 25 µM CdCl₂ and above, and analysis of different cell death pathways indicated the presence of necrosis after treatment with 50 µM CdCl₂. At the molecular level, we observed an increase in the protective protein heat shock protein 70 (HSP70), starting at 10 µM CdCl₂. At the barrier level, transepithelial electrical resistance (TEER) decreased while paracellular permeability (PCP) significantly increased after the treatment, showing an EC₅₀ of 6 and 16 µM CdCl₂, respectively, and indicating the loss of barrier integrity. In conclusion, our data reveal that CdCl₂ toxicity in Caco-2 cells can be detected at the barrier level at very low concentrations; also, HSP70 was shown to be a sensitive marker for detecting in vitro cadmium-induced toxicity.     

Transformation of prostatic epithelial cells and fibroblasts with cadmium chloride in vitro

Primary cultures of fibroblasts and epithelial cells were established from rat ventral prostate (RVP), canine (CP), baboon (BP), and human (HP) prostates, and were used in an assay system to evaluate cadmium chloride (CdCl₂) cytotoxicity in vitro. Fibroblasts were always more susceptible to CdCl₂ cytotoxicity than the epithelial cells of the same species. There was a distinct species variability to CdCl₂ cytotoxicity, with RVP cells being greater than 200 times more susceptible than HP. Primary cultures treated with CdCl₂ were subcultivated to establish cell lines. Only RVP fibroblast and epithelial cells resulted in permanent cell lines. Two fibroblast and two epithelial cell lines were derived from CdCl₂-treated RVP cell cultures. The epithelial cell lines possessed tonofilaments, desmosomes and keratin. All four cell lines were resistant to CdCl₂, had different karyotypes and an excess of chromosome 13. These results demonstrate the transforming potential of cadmium on prostate cells. The role of metallothionein and the significance of extra chromosomes 13 are discussed as possible factors of cadmium resistance.     

Oncogenicity of rat prostate cells transformed in vitro with cadmium chloride

Three rat ventral prostate (RVP) cell lines transformed after in vitro treatment with cadmium chloride (CdCl₂) and one control untreated cell line were tested for tumorigenicity in newborn rats. All three cadmium-transformed RVP cell lines induced tumors at the site of inoculation in 95–100% of animals. The fibroblastoid RVP56Cd cell line induced sarcomas, whereas the epithelial cell lines RVP47-3G and RVP47-3F produced highly differentiated squamous cell carcinomas. About 20% of animals injected with RVP47-3G developed lung and splenic metastases. The tumors could be further passaged into young rats. The sarcomas had a hyperdiploid modal chromosome number similar to that of the RVP56Cd cell line. Carcinomas induced by the RVP47-3G cell line had a large proportion of stromal metaphases. The modal chromosome number of these carcinomas was in the hypertriploid-hypotetraploid range, similar to that of the parental cell line. These results demonstrate that treatment of RVP cells with CdCl₂ in vitro results in neoplastic transformation. Since both fibroblastoid and epithelial prostate cells have undergone transformation, it seems possible that cadmium acted as a carcinogen without cell specificity. The susceptibility of these cells to the carcinogenic effect may be related to their resistance to cadmium. In the process of neoplastic transformation induced by CdCl₂ in RVP epithelial cells changes of squamous metaplasia occur, and probably precede acquisition of tumorigenicity.This work was supported in part by an American Cancer Society Institutional Grant     

Action of ricin and its polypeptide chains on cultured hepatoma cells

The effect of ricin, the glycoproteic toxin from castor beans, as well as of its two polypeptide chains A and B, was studied on hepatoma tissue cell suspension cultures (HTC). The isolated A and B chains have no action. Ricin at a 0.3 g/ml concentration, for 3×10⁵ cells/ml, has a cytostatic effect which becomes cytotoxic after 72 h. This effect exists even if the cells are preincubated for 4 h with ricin and washed afterwards. The protein content of the ricin-treated cells decreases. Protein synthesis measured by the incorporation of [³H]-leucine in the cell proteins begins to be inhibited after 30 min and is completely blocked after 2 h for a ricin concentration of 0.3 g/ml. DNA synthesis is inhibited from 1 h on. Finally the level of the DNA slows down from 3 h on, whereas the level of RNA abruptly falls between 4 and 5 h. The inhibitory effects are likely to be a consequence of protein synthesis inhibition, but a direct effect on the nucleic acids is not excluded.     

齐墩果酸诱导人肝癌HepG2细胞凋亡及其作用机制研究

目的探讨齐墩果酸对人肝癌HepG2细胞增殖的抑制作用和诱导凋亡作用,并考察其作用机制。方法采用MTT法检测齐墩果酸对HepG2细胞增殖的影响,吖啶橙/溴化乙锭(AO/EB)双重染色法进行细胞形态学观察,流式细胞仪检测细胞凋亡、活性氧(ROS)水平和线粒体膜电位(MMP)的变化。结果随着齐墩果酸浓度的升高,抑制率显著增加,呈现明显的时间与剂量相关性;齐墩果酸在25.0、50.0、100.0μmol/L作用12 h时,细胞形态学发生改变,细胞的总凋亡率随着齐墩果酸浓度的增加而升高,细胞中ROS水平随着齐墩果酸浓度的增加而升高,细胞内的MMP水平随着齐墩果酸浓度的增加而降低。结论齐墩果酸通过调节ROS和MMP体外抑制人肝癌HepG2细胞的增殖和诱导其凋亡     

Cerium oxide nanoparticles protects against acrylamide induced toxicity in HepG2 cells through modulation of oxidative stress

Acrylamide (AA) is a toxic chemical compound found in cooked foods. Considerable evidences suggest that oxidative stress and mitochondrial dysfunction are contributed to AA toxicity. Ceric oxide (CeO₂) nanoparticles (nano-ceria) have the potential to be developed as a therapeutic for oxidative stress insults due to their catalytic antioxidant properties. In this study we investigated, whether nano-ceria exerted a protective effect against AA-induced cytotoxicity and oxidative damage. HepG2 human cancer cell lines were exposed to nano-ceria (50, 100, and 200?µM) and after 30?min, AA in the half maximal inhibitory concentration (IC50) concentration (200?µM) was added to the cells. Twenty four hours later, cellular viability, reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and cellular levels of glutathione (GSH) were assayed. AA decreased cell viability and pretreatment with nano-ceria significantly decreased AA-induced cytotoxicity. In addition, nano-ceria alleviated AA-induced ROS generation and LPO and depressed GSH level. Our results suggested that nano-ceria prevented cellular and oxidative damage induced by AA.     

Cytotoxic effects of procyanidins from Castanea mollissima Bl. shell on human hepatoma G2 cells in vitro

Significant cytotoxic effects of procynadins from chestnut (Castanea mollissima Bl.) shell (CSPC) on human hepatoma G2 (HepG2) cells were found in vitro. CSPC could inbibit HepG2 proliferation in a dose-dependent manner (100–400 μg/mL), arrest cell cycle in the G₀/G₁ phase, induce apoptosis and trigger necrosis of HepG2. Proapoptotic effect of CSPC was evidenced by nuclear condensation, internucleosomal DNA fragmentation. Treatment of HepG2 cells with CSPC caused a loss of mitochondrial membrane potential and stimulated reactive oxidative species (ROS) generation. These results suggested CSPC could trigger apoptosis and necrotic cell death in HepG2 cell, which might be associated with ROS generation through the mitochondria-dependent signaling way.     

Genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells

The recent finding that acrylamide (AA), a carcinogen in animal experiments and a probable human carcinogen, is formed in foods during cooking raises human health concerns. The relevance of dietary exposure for humans is still under debate. The purpose of the study was to evaluate the possible genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells, a cell line of great relevance to detect genotoxic/antigenotoxic substances, using single cell gel electrophoresis (SCGE) assay and micronucleus test (MNT). In order to clarify the underlying mechanism(s) we evaluated the intracellular generation of reactive oxygen species (ROS) and the level of oxidative DNA damage by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The involvement of glutathione (GSH) in the AA-induced oxidative stress was examined through treatment with buthionine sulfoximine (BSO) to deplete GSH. The results indicate that AA caused DNA strand breaks and increase in frequency of MN in HepG2 cells in a dose-dependent manner. The possible mechanism underlies the increased levels of ROS, depletion of GSH and increase of 8-OHdG formation in HepG2 cells treated with AA. We conclude that AA exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.     

The role of CYP3A4 in amiodarone-associated toxicity on HepG2 cells

Amiodarone is a class III antiarrhythmic drug with potentially life-threatening hepatotoxicity. Recent in vitro investigations suggested that the mono-N-desethyl (MDEA) and di-N-desethyl (DDEA) metabolites may cause amiodarone's hepatotoxicity. Since cytochrome P450 (CYP) 3A4 is responsible for amiodarone N-deethylation, CYP3A4 induction may represent a risk factor. Our aim was therefore to investigate the role of CYP3A4 in amiodarone-associated hepatotoxicity. First, we showed that 50 μM amiodarone is more toxic to primary human hepatocytes after CYP induction with rifampicin. Second, we overexpressed human CYP3A4 in HepG2 cells (HepG2 cells/CYP3A4) for studying the interaction between CYP3A4 and amiodarone in more detail. We also used HepG2 wild type cells (HepG2 cells/wt) co-incubated with human CYP3A4 supersomes for amiodarone activation (HepG2 cells/CYP3A4 supersomes). Amiodarone (10–50 μM) was cytotoxic for HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes, but not for HepG2 cells/wt or less toxic for HepG2 cells/wt incubated with control supersomes without CYP3A4. Co-incubation with ketoconazole, attenuated cytotoxicity of amiodarone incubated with HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes. MDEA and DDEA were formed only in incubations containing HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes but not by HepG2 cells/wt or HepG2 cells/wt with control supersomes. Metabolized amiodarone triggered the production of reactive oxygen species, induced mitochondrial damage and cytochrome c release, and promoted apoptosis/necrosis in HepG2 cells/CYP3A4, but not HepG2 cells/wt. This study supports the hypothesis that a high CYP3A4 activity is a risk factor for amiodarone's hepatotoxicity. Since CYP3A4 inducers are used frequently and amiodarone-associated hepatotoxicity can be fatal, our observations may be clinically relevant.     

沙蟾毒精抑制肝癌HepG2细胞黏附、迁移和侵袭的作用

目的研究沙蟾毒精(Arenobufagin)对人肝癌细胞HepG2黏附、迁移和侵袭的抑制作用。方法 MTT法检测沙蟾毒精对HepG2细胞活力和细胞黏附能力的影响,划痕实验观察沙蟾毒精对HepG2细胞运动能力的影响,Transwell小室模型研究沙蟾毒精对HepG2细胞的迁移和侵袭的抑制作用,Western blot检测基质金属蛋白酶MMP 2和MMP 9的表达水平变化。结果与空白组相比,沙蟾毒精可降低HepG2细胞的黏附能力,使Transwell小室膜上的肝癌细胞明显减少,并且呈剂量依赖性。Western blot结果显示沙蟾毒精可以明显抑制基质金属蛋白酶MMP 2和MMP 9的表达。结论沙蟾毒精能抑制人肝癌细胞HepG2的黏附、迁移和侵袭,其机制可能与抑制MMP 2和MMP 9的表达有关。     

Sulfur amino acid metabolism in doxorubicin-resistant breast cancer cells

Although methionine dependency is a phenotypic characteristic of tumor cells, it remains to be determined whether changes in sulfur amino acid metabolism occur in cancer cells resistant to chemotherapeutic medications. We compared expression/activity of sulfur amino acid metabolizing enzymes and cellular levels of sulfur amino acids and their metabolites between normal MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Adr) cells. The S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in MCF-7/Adr cells decreased to ~ 10% relative to that in MCF-7 cells, which may have resulted from down-regulation of S-adenosylhomocysteine hydrolase. Expression of homocysteine-clearing enzymes, such as cystathionine beta-synthase, methionine synthase/methylene tetrahydrofolate reductase, and betaine homocysteine methyltransferase, was up-regulated in MCF-7/Adr cells, suggesting that acquiring doxorubicin resistance attenuated methionine-dependence and activated transsulfuration from methionine to cysteine. Homocysteine was similar, which is associated with a balance between the increased expressions of homocysteine-clearing enzymes and decreased extracellular homocysteine. Despite an elevation in cysteine, cellular GSH decreased in MCF-7/Adr cells, which was attributed to over-efflux of GSH into the medium and down-regulation of the GSH synthesis enzyme. Consequently, MCF-7/Adr cells were more sensitive to the oxidative stress induced by bleomycin and menadione than MCF-7 cells. In conclusion, our results suggest that regulating sulfur amino acid metabolism may be a possible therapeutic target for chemoresistant cancer cells. These results warrant further investigations to determine the role of sulfur amino acid metabolism in acquiring anticancer drug resistance in cancer cells using chemical and biological regulators involved in sulfur amino acid metabolism.     

大黄素诱导人肝癌HepG2细胞线粒体凋亡作用研究

目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧（ROS）含量、Ca²⁺浓度、线粒体膜电位（MMP）变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度（IC₅₀）为（77.42±1.25）μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca²⁺浓度明显增加（P<0.05、0.01、0.001）,80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降（P<0.05、0.01、0.001）。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。     

In vitro toxicity assessment of nanocrystals in tissue‐type cells and macrophage cells

Nanocrystals (NCs), a type of innovative material particle, are a potential drug delivery platform that aims to improve the bioavailability of hydrophobic drugs. However, due to the lack of consideration of their toxicity, existing studies have not investigated whether the nanoscale properties of NCs, such as particle sizes, may lead to NC‐induced toxicity. Because of the disparity between the rapid development of NCs and the lack of studies regarding NC toxicity, the present study investigated possible NC toxicity and clarified the relationship between particle sizes and NC toxicity. RAW264.7 and HepG2 cells were chosen as representatives of macrophage cells and tissue‐type cells, respectively. Monosodium urate NCs were used as a drug model. Different particle sizes of monosodium urate NCs were prepared using precipitation methods. Methyl tetrazolium, lactate dehydrogenase, oxidative stress and apoptosis/necrosis assays were then used to evaluate cell damage and recovery. The results showed that small NC particle sizes produced higher toxicity than larger ones. In immune cells, these cytotoxic effects were greater than in tissue cells. After removal of small NCs, tissue cell damage could be significantly reversed, while immune cells were only slightly restored. However, after removal of large NCs, both cell types had almost no recovery. In summary, despite conventional wisdom, our research confirmed that NCs are not very safe and that NC particle sizes are closely related to the degree of NC toxicity.