Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay

Titanium dioxide nanoparticles (TiO₂‐NPs) are being used increasingly for various industrial and consumer products, including cosmetics and sunscreens because of their photoactive properties. Therefore, the toxicity of TiO₂‐NPs needs to be thoroughly understood. In the present study, the genotoxicity of 10nm uncoated sphere TiO₂‐NPs with an anatase crystalline structure, which has been well characterized in a previous study, was assessed using the Salmonella reverse mutation assay (Ames test) and the single‐cell gel electrophoresis (Comet) assay. For the Ames test, Salmonella strains TA102, TA100, TA1537, TA98 and TA1535 were preincubated with eight different concentrations of the TiO₂‐NPs for 4 h at 37 °C, ranging from 0 to 4915.2 µg per plate. No mutation induction was found. Analyses with transmission electron microscopy (TEM) and energy‐dispersive X‐ray spectroscopy (EDS) showed that the TiO₂‐NPs were not able to enter the bacterial cell. For the Comet assay, TK6 cells were treated with 0–200 µg ml^–1 TiO₂‐NPs for 24 h at 37 °C to detect DNA damage. Although the TK6 cells did take up TiO₂‐NPs, no significant induction of DNA breakage or oxidative DNA damage was observed in the treated cells using the standard alkaline Comet assay and the endonuclease III (EndoIII) and human 8‐hydroxyguanine DNA‐glycosylase (hOGG1)‐modified Comet assay, respectively. These results suggest that TiO₂‐NPs are not genotoxic under the conditions of the Ames test and Comet assay. Published 2012. This article is a US Government work and is in the public domain in the USA.     

Genotoxicity of Aspartame

In the present study, the genotoxic effects of the low‐calorie sweetener aspartame (ASP), which is a dipeptide derivative, was investigated using chromosome aberration (CA) test, sister chromatid exchange (SCE) test, micronucleus test in human lymphocytes and also Ames/Salmonella/ microsome test. ASP induced CAs at all concentrations (500, 1000 and 2000 µg/ml) and treatment periods (24 and 48 h) dose‐dependently, while it did not induce SCEs. On the other hand, ASP decreased the replication index (RI) only at the highest concentration for 48 h treatment period. However, ASP decreased the mitotic index (MI) at all concentrations and treatment periods dose‐dependently. In addition, ASP induced micronuclei at the highest concentrations only. This induction was also dose‐dependent for 48 hours treatment period. ASP was not mutagenic for Salmonella typhimurium TA98 and TA100 strains in the absence and presence of S9 mix.     

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO₂) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO₂ NPs. The purpose of this study is to evaluate the genotoxicity of TiO₂ NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO₂ NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200 mg/kg body weight every day for 30 days. The γ-H₂AX assay showed TiO₂ NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO₂ NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO₂ NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24 h and 48 h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24 h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2 h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO₂ NPs, and TiO₂ NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO₂ NPs induced genotoxic effects.     

Mutagenicity testing of quinine with submammalian and mammalian systems

Quinine hydrochloride was assayed for genotoxic activity by using 4 different test systems with distinct genetic endpoints. No indications for point mutations were observed in the Ames system. In 3 cytogenetic tests performed on small rodents, Chinese hamsters showed no genotoxic activity, while inbred strains of mice revealed a dose dependent increase of SCEs, enhanced incidence of micronuclei and elevated chromatid breaks.     

The immunomodulatory effects of titanium dioxide and silver nanoparticles

Due to their characteristic physical, chemical and optical properties, titanium dioxide and silver nanoparticles are attractive tools for use in a wide range of applications. The use of nanoparticles for biological applications is, however, dependent upon their biocompatibility with living cells. Because of the importance of inflammation as a modulator of human health, the safe and efficacious in vivo use of titanium dioxide and silver nanoparticles is inherently linked to a favorable interaction with immune system cells. However, both titanium dioxide and silver nanoparticles have demonstrated potential to exert immunomodulatory and immunotoxic effects. Titanium dioxide and silver nanoparticles are readily internalized by immune system cells, may accumulate in peripheral lymphoid organs, and can influence multiple manifestations of immune cell activity. Although the factors influencing the biocompatibility of titanium dioxide and silver nanoparticles with immune system cells have not been fully elucidated, nanoparticle core composition, size, concentration and the duration of cell exposure seem to be important. Because titanium dioxide and silver nanoparticles are widely utilized in pharmaceutical, commercial and industrial products, it is vital that their effects on human health and immune system function be more thoroughly evaluated.     

Effects of titanium dioxide nanoparticles on human keratinocytes

Titanium dioxide (TiO₂) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO₂ inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO₂ nanoparticles (H₂TiO₇) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO₂ particles varying in size (Fine, Ultrafine and H₂TiO₇) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO₂; however, there is no consistent effect on cell viability and proliferation with either of these TiO₂ particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO₂, we did not observe any significant effect of UV-C exposure combined with TiO₂ treatment on HaCaTs. Furthermore, TiO₂-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H₂TiO₇ nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO₂.     

Influence of silver and titanium dioxide nanoparticles on in vitro blood-brain barrier permeability

An in vitro blood-brain barrier (BBB) model being composed of co-culture with endothelial (bEnd.3) and astrocyte-like (ALT) cells was established to evaluate the toxicity and permeability of Ag nanoparticles (AgNPs; 8 nm) and TiO₂ nanoparticles (TiO₂NPs; 6 nm and 35 nm) in normal and inflammatory central nervous system. Lipopolysaccharide (LPS) was pre-treated to simulate the inflammatory responses. Both AgNPs and Ag ions can decrease transendothelial electrical resistance (TEER) value, and cause discontinuous tight junction proteins (claudin-5 and zonula occludens-1) of BBB. However, only the Ag ions induced inflammatory cytokines to release, and had less cell-to-cell permeability than AgNPs, which indicated that the toxicity of AgNPs was distinct from Ag ions. LPS itself disrupted BBB, while co-treatment with AgNPs and LPS dramatically enhanced the disruption and permeability coefficient. On the other hand, TiO₂NPs exposure increased BBB penetration by size, and disrupted tight junction proteins without size dependence, and many of TiO₂NPs accumulated in the endothelial cells were observed. This study provided the new insight of toxic potency of AgNPs and TiO₂NPs in BBB.     

Cell uptake and oral absorption of titanium dioxide nanoparticles

Large efforts are invested on the development of in vitro tests to evaluate nanomaterial (NM) toxicity. In order to assess the relevance of the adverse effects identified in in vitro toxicity tests a thorough understanding of the biokinetics of NMs is critical. We used different in vitro and in vivo test methods to evaluate cell uptake and oral absorption of titanium dioxide nanoparticles (TiO₂ NPs). These NPs were readily uptaken by A549 cells (carcinomic human alveolar basal epithelial cells) in vitro. Such rapid uptake contrasted with a very low oral absorption in a differentiated Caco-2 monolayer system (human epithelial colorectal adenocarcinoma cells) and after oral gavage administration to rats. In this oral study, no significant increase in the levels of titanium was recorded by ICP-MS in any of the tissues evaluated (including among other: small intestine, Peyer's patches, mesenteric lymph nodes, liver, and spleen). No NPs were observed by TEM in sections of the small intestine, except for several particles in the cytoplasm of a cell from a Peyer's Patch area. The observation of NPs in Peyer's Patch suggests that the Caco-2 monolayer system is likely to underestimate the potential for oral absorption of NPs and that the model could be improved by including M-cells in co-culture.     

Comparative study on effects of two different types of titanium dioxide nanoparticles on human neuronal cells

Titanium dioxide (TiO₂) are among most frequently used nanoparticles (NPs). They are present in a variety of consumer products, including food industry in which they are employed as an additive. The potential toxic effects of these NPs on mammal cells have been extensively studied. However, studies regarding neurotoxicity and specific effects on neuronal systems are very scarce and, to our knowledge, no studies on human neuronal cells have been reported so far. Therefore, the main objective of this work was to investigate the effects of two types of TiO₂ NPs, with different crystalline structure, on human SHSY5Y neuronal cells. After NPs characterization, a battery of assays was performed to evaluate the viability, cytotoxicity, genotoxicity and oxidative damage in TiO₂ NP-exposed SHSY5Y cells. Results obtained showed that the behaviour of both types of NPs resulted quite comparable. They did not reduce the viability of neuronal cells but were effectively internalized by the cells and induced dose-dependent cell cycle alterations, apoptosis by intrinsic pathway, and genotoxicity not related with double strand break production. Furthermore, all these effects were not associated with oxidative damage production and, consequently, further investigations on the specific mechanisms underlying the effects observed in this study are required.     

Induction of chronic inflammation in mice treated with titanium dioxide nanoparticles by intratracheal instillation

Titanium dioxide nanoparticles (TNP) are nanomaterials which have various applications including photocatalysts, cosmetics, and pharmaceuticals because of their high stability, anticorrosiveness, and photocatalytic properties. Induction of cytokines and potential chronic inflammation were investigated in mice treated with TNP (5 mg/kg, 20 mg/kg, and 50 mg/kg) by a single intratracheal instillation. Pro-inflammatory cytokines such as IL-1, TNF-a, and IL-6 were significantly induced in a dose-dependent manner at day 1 after instillation. The levels of Th1-type cytokines (IL-12 and IFN-γ) and Th2-type cytokines (IL4, IL-5 and IL-10) were also elevated dose-dependently at day 1 and the inflammatory responses were sustained until the remainder of experimental period for 14 days. By the induction of Th2-type cytokines, the increased B cell distributions both in spleen and in blood, and increased IgE production in BAL fluid and serum were observed. In lung tissue, increase of inflammatory proteins (MIP and MCP) and granuloma formation were observed. Furthermore, the expressions of genes related with antigen presentation (H2-T23, H2-T17, H2-K1, and H2-Eb1) and genes related with the induction of chemotaxis of immune cells (Ccl7, Ccl3, Cxcl1, Ccl4, Ccl2) were markedly increased using microarray analysis. From these data, it could be suggested that TNP possibly cause chronic inflammatory diseases through Th2-mediated pathway in mice.     

Titanium dioxide nanoparticles induced intracellular calcium homeostasis modification in primary human keratinocytes. Towards an in vitro explanation of titanium dioxide nanoparticles toxicity

Abstract

Deciphering the molecular basis of toxicology mechanism induced by nanoparticles (NPs) remains an essential challenge. Ion Beam Analysis (IBA) was applied in combination with Transmission Electron Microscopy and Confocal Microscopy to analyze human keratinocytes exposed to TiO₂-NPs. Investigating chemical elemental distributions using IBA gives rise to a fine quantification of the TiO₂-NPs uptake within a cell and to the determination of the intracellular chemical modifications after TiO₂-NPs internalization. In addition, fluorescent dye-modified TiO₂-NPs have been synthesized to allow their detection, precise quantification and tracking in vitro. The internalization of these TiO₂-NPs altered the calcium homeostasis and induced a decrease in cell proliferation associated with an early keratinocyte differentiation, without any indication of cell death. Additionally, the relation between the surface chemistry of the TiO₂-NPs and their in vitro toxicity is clearly established and emphasizes the importance of the calcium homeostasis alteration in response to the presence of TiO₂-NPs.     

Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on fish cells in vitro

Intrinsic genotoxic and cytotoxic potential of titanium dioxide (TiO2) engineered nanoparticles (ENPs) were evaluated in a metabolically competent, established fish cell line derived from rainbow trout (Oncorhyncus mykiss) gonadal tissue (i.e. RTG-2 cells). Prior to evaluation of the toxic potential, mean size of the ENPs was determined using transmission electron microscopy (TEM). As a prerequisite, an extensive characterisation of the ENPs was carried out following sonication which enabled the synthesis of an efficient dosing strategy for the cells in which exposure in phosphate buffered saline (PBS) gave an optimal agglomeration effects compared to distilled water (H2O) and minimal essential media (MEM). Interaction of the ENPs with cells under scanning electron microscope (SEM) was also studied. The genotoxic and cytotoxic potential of the ENPs were determined either alone or in combination with ultraviolet radiation (i.e. UVA). Whilst genotoxic potential was determined by evaluating DNA strand breaks using single cell gel electrophoresis (SCGE) or the comet assay and induction of cytogenetic damage using cytokinesis-blocked micronucleus (MN) assay, cytotoxicity was determined by measuring the retention of supra vital stain, neutral red, by the lysosomes using the neutral red retention (NRR) assay. In addition, while performing the comet assay, lesion specific bacterial endonuclease, formamidopyrimidine DNA glycosylase (Fpg), which recognises oxidised purine bases, was used to determine oxidative DNA damage. The results suggested that the highest concentration of the ENPs (i.e. 50 microg ml(-1)) did not produce elevations in DNA damage over 4 h (comet assay), 24 h (modified comet assay) or 48 h (MN assay) exposures in the absence of UVA irradiation, although there was a significant reduction in lysosomal integrity over 24 h exposure (NRR assay). The induction of MN did not show any enhanced levels as a function of ENP concentration. A significantly increased level of strand breaks was observed in combination with UVA (3 kJ m(-2)). In general, the NRR assay suggested elevated levels of cytotoxicity when the UVA exposure was carried out with MEM compared to PBS, although both showed an increase when in combination with the highest concentration of ENPs (i.e. 50 microg ml(-1)). Overall, the study emphasises the need for adoption of an holistic approach while evaluating the potential toxic effects of ENPs in which appropriate measures should be taken to avoid agglomeration or aggregation to facilitate efficient cellular uptake to evaluate potential biological responses.     

体内基因突变试验研究进展

遗传毒性研究是新药非临床安全性评价的重要内容之一。由于遗传毒性体外细胞试验不能完全反映整体动物系统对受试物的吸收、分布、代谢和排泄等情况,而且试验结果的假阳性率过高,因此采用体外细胞试验进行药物安全性评价尚存在一定局限性。为了进一步完善对致突变风险的控制,尚需建立更加灵敏的体内基因突变试验方法。本文主要就体内基因突变试验的基本原理、优缺点及基本应用等研究进展进行综述,并对这些体内试验的发展和完善提出一些展望。     

Toxicity assessment of six titanium dioxide nanoparticles in human epidermal keratinocytes

Purpose: The aim of this study was to evaluate and compare the toxicity of six different types of titanium dioxide (TiO₂) nanoparticles (NP) on human epidermal keratinocytes (HEK).

Materials and methods: Six TiO₂ NP (A (10?nm), A*(32?nm), B (27.5?nm), C (200?nm), C*(30–40?nm), and D*(200–400?nm)) were suspended in water or culture medium and characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). In addition, these NP were assayed with cell viability, cytokine release and cellular uptake in HEK.

Results: TiO₂NP did not change in shape in the culture medium when visualized by TEM. There was an increase in agglomeration with all TiO₂NP in the medium when measured by DLS. Since TiO₂NP interfered with the CellTiter 96®AQueous One and MTT assays but had a minimal effect on alamar Blue (aB). The aB viability assay was selected to assess all six types of TiO₂NP and sample B had a statistically significant decrease in viability at 0.4?mg/ml. A slight increase in TNF-α was noted in sample A*, C, and D* at as low as 0.05?mg/ml. Sample A* and B at certain concentrations showed an increase in Interleukin (IL)-6. IL-10 and IL-1β release for all TiO₂NP were noted around the detection limit with no significant changes compared to control. A statistically significant decrease in IL-8 was noted for all TiO₂NP at the highest concentrations due to the adsorption of IL-8 by TiO₂. All TiO₂NP were localized within cytoplasmic vacuoles of HEK and the element Ti was detected by energy-dispersive x-ray spectroscopy analysis.

Conclusions: Based on cell viability, only sample B was slightly cytotoxic to HEK and samples B and A* have the potential to cause inflammation indicated by an increase in IL-6.     

In vivo genotoxicity study of titanium dioxide nanoparticles using comet assay following intratracheal instillation in rats

Titanium dioxide (TiO₂) is widely used as a white pigment in paints, plastics, inks, paper, creams, cosmetics, drugs and foods. In the present study, the genotoxicity of anatase TiO₂ nanoparticles was evaluated in vivo using the comet assay after a single or repeated intratracheal instillation in rats. The nanoparticles were instilled intratracheally at a dosage of 1.0 or 5.0 mg/kg body weight (single instillation group) and 0.2 or 1.0 mg/kg body weight once a week for 5 weeks (repeated instillation group) into male Sprague–Dawley rats. A positive control, ethyl methanesulfonate (EMS) at 500 mg/kg, was administered orally 3 h prior to dissection. Histopathologically, macrophages and neutrophils were detected in the alveolus of the lung in the 1.0 and 5.0 mg/kg TiO₂ groups. In the comet assay, there was no increase in % tail DNA in any of the TiO₂ groups. In the EMS group, there was a significant increase in % tail DNA compared with the negative control group. TiO₂ nanoparticles in the anatase crystal phase are not genotoxic following intratracheal instillation in rats.     

Biomarkers of environmental contaminants in field population of green mussel (Perna viridis) from Karnataka–Kerala coast (South West coast of India)

The green mussel Perna viridis was sampled from relatively clean and contaminated sites along the Kartanata–Kerala coast (south west coast of India) to study the tissue concentration of trace metals and biological responses to stress (biomarkers) such as sister chromatid exchange (SCE), chromosomal aberration, micronucleus (MN) test, hemic neoplasia (HN), Chromotest (Ames test) and comet assay. In general, mean tissue concentrations of toxic trace metals collected from 25 sampling sites were found to be below the World Health Organisation (WHO) permissible concentration given for seafood. The digestive gland extract of mussels from all 25 sampling sites showed negative reaction for mutagenic activity (Ames test) in the absence of metabolic activation. Very low levels of chromosomal aberration, SCE, MN, HN and comet cells were observed in mussels collected from both the urban associated and relatively clean sites. This study seems to indicate that that the coastal waters of Karnataka and Kerala are minimally contaminated with genotoxic and carcinogenic chemicals.Special Issue on Biomarkers of Marine Pollution and Bioremediation     

Genotoxicity evaluation of nanosized titanium dioxide,synthetic amorphous silica and multi-walled carbon nanotubes in human lymphocytes

Toxicological characterization of manufactured nanomaterials (NMs) is essential for safety assessment, while keeping pace with innovation from their development and application in consumer products. The specific physicochemical properties of NMs, including size and morphology, might influence their toxicity and have impact on human health. The present work aimed to evaluate the genotoxicity of nanosized titanium dioxide (TiO₂), synthetic amorphous silica (SAS) and multiwalled carbon nanotubes (MWCNTs), in human lymphocytes. The morphology and size of those NMs were characterized by transmission electron microscopy, while the hydrodynamic particle size-distributions were determined by dynamic light scattering. Using a standardized procedure to ensure the dispersion of the NMs and the cytokinesis-block micronucleus assay (without metabolic activation), we observed significant increases in the frequencies of micronucleated binucleated cells (MNBCs) for some TiO₂ NMs and for two MWCNTs, although no clear dose–response relationships could be disclosed. In contrast, all forms of SAS analyzed in this study were unable to induce micronuclei. The present findings increase the weight of evidence towards a genotoxic effect of some forms of TiO₂ and some MWCNTs. Regarding safety assessment, the differential genotoxicity observed for closely related NMs highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs.     

In situ quantification of diverse titanium dioxide nanoparticles unveils selective endoplasmic reticulum stress-dependent toxicity

Although titanium dioxide nanoparticles (TiO₂ NPs) have been extensively studied, their possible impact on health due to their specific properties supported by their size and geometry, remains to be fully characterized to support risk assessment. To further document NPs biological effects, we investigated the impact of TiO₂ NPs morphology on biological outcomes. To this end, TiO₂ NPs were synthesized as nanoneedles (NNs), titanate scrolled nanosheets (TNs), gel-sol-based isotropic nanoparticles (INPs) and tested for perturbation of cellular homeostasis (cellular ion content, cell proliferation, stress pathways) in three cell types and compared to the P25. We showed that TiO₂ NPs were internalized at various degrees and their toxicity depended on both titanium content and NPs shape, which impacted on intracellular calcium homeostasis thereby leading to endoplasmic reticulum stress. Finally, we showed that a minimal intracellular content of TiO₂ NPs was mandatory to induce toxicity enlightening once more the crucial notion of internalized dose threshold beside the well-recognized dose of exposure.     

Evaluation of cytogenotoxicity and oxidative stress parameters in male Swiss mice co-exposed to titanium dioxide and zinc oxide nanoparticles

A number of studies have investigated the adverse toxic effects of titanium dioxide (TiO₂) nanoparticles (NPs) or zinc oxide (ZnO) NPs. Information on the potential genotoxic effects of the interactions of TiO₂ NPs and ZnO NPs in vivo is lacking. Therefore, this study was designed to investigate the cytogenotoxicity of TiO₂ NPs or ZnO NPs alone or their mixtures using the bone marrow micronucleus assay, and mechanism of damage through the evaluation of oxidative stress parameters in the liver and kidney tissues of Swiss mice. Intraperitoneal administration of doses between 9.38 and 150.00 mg/kg of TiO₂ NPs or ZnO NPs or TiO₂ NPs + ZnO NPs was performed for 5 and 10 days, respectively. TiO₂ NPs alone induced a significant (P < 0.05) increase in micronucleated (Mn) polychromatic erythrocytes (PCEs) at the applied doses compared with the negative controls, with a significant difference between 5 and 10 days for TiO₂ NPs alone and TiO₂ NPs + ZnO NPs. Concurrently, TiO₂ NPs alone for 5 days and TiO₂ NPs and TiO₂ NPs + ZnO NPs for 10 days significantly (P < 0.05) decreased the percentage PCE: normochromatic erythrocyte (NCE) indicating cytotoxicity; with a significant difference between the two periods. Significant (P < 0.001) changes in the activities of superoxide dismutase (SOD) and catalase (CAT), and levels of reduced glutathione (GSH) and malondialdehyde (MDA) were observed in the liver and kidney of mice exposed to TiO₂ NPs or ZnO NPs alone or their mixtures. These results suggest that TiO₂ NPs alone was genotoxic; TiO₂ NPs and TiO₂ NPs + ZnO NPs were noticeably cytotoxic while ZnO NPs was not cytogenotoxic. The individual NPs or their mixtures induced oxidative stress.