Amplification of arsenic genotoxicity by TiO2 nanoparticles in mammalian cells: new insights from physicochemical interactions and mitochondria

Titanium dioxide nanoparticles (TiO₂ NPs) have shown great adsorption capacity for arsenic (As); however, the potential impact of TiO₂ NPs on the behavior and toxic responses of As remains largely unexplored. In the present study, we focused on the physicochemical interaction between TiO₂ NPs and As(III) to clarify the underlying mechanisms involved in their synergistic genotoxic effect on mammalian cells. Our data showed that As(III) mainly interacted with TiO₂ NPs by competitively occupying the sites of hydroxyl groups on the surface of TiO₂ NP aggregates, resulting in more aggregation of TiO₂ NPs. Although TiO₂ NPs at concentrations used here had no cytotoxic or genotoxic effects on cells, they efficiently increased the genotoxicity of As(III) in human-hamster hybrid (A_L) cells. The synergistic genotoxicity of TiO₂ NPs and As(III) was partially inhibited by various endocytosis pathway inhibitors while it was completely blocked by an As(III)-specific chelator. Using a mitochondrial membrane potential fluorescence probe, a reactive oxygen species (ROS) probe together with mitochondrial DNA-depleted ρ⁰ A_L cells, we discovered that mitochondria were essential for mediating the synergistic DNA-damaging effects of TiO₂ NPs and As(III). These data provide novel mechanistic proof that TiO₂ NPs enhanced the genotoxicity of As(III) via physicochemical interactions, which were mediated by mitochondria-dependent ROS.     

Activation of the inflammasome by amorphous silica and TiO2 nanoparticles in murine dendritic cells

Abstract

Nanomaterials are increasingly used in various food applications. In particular, nanoparticulate amorphous SiO₂ is already contained, e.g., in spices. Since intestinal dendritic cells (DC) could be critical targets for ingested particles, we compared the in vitro effects of amorphous silica nanoparticles with fine crystalline silica, and micron-sized with nano-sized TiO₂ particles on DC. TiO₂- and SiO₂-nanoparticles, as well as crystalline silica led to an upregulation of MHC-II, CD80, and CD86 on DC. Furthermore, these particles activated the inflammasome, leading to significant IL-1β-secretion in wild-type (WT) but not Caspase-1- or NLRP3-deficient mice. Silica nanoparticles and crystalline silica induced apoptosis, while TiO₂ nanoparticles led to enhanced production of reactive oxygen species (ROS). Since amorphous silica and TiO₂ nanoparticles had strong effects on the activation-status of DC, we suggest that nanoparticles, used as food additives, should be intensively studied in vitro and in vivo, to ensure their safety for the consumer.     

Acute and chronic toxicity of nano-scale TiO2 particles to freshwater fish,cladocerans, and green algae,and effects of organic and inorganic substrate on TiO2 toxicity

This research evaluated the toxicity of TiO₂ nanoparticles to freshwater aquatic organisms and the effects of organic and inorganic material on TiO₂ toxicity. The fathead minnow was much less acutely sensitive to TiO₂ (LC50 500 mg/l and higher) than Ceriodaphnia dubia and Daphnia pulex (mean LC50 values 7.6 and 9.2 mg/l, respectively). Total organic carbon levels of 1.5 mg/l decreased TiO₂ acute toxicity to C. dubia (LC50 > 100 mg/l), but kaolinite clay decreased TiO₂ toxicity to a lesser extent. In chronic toxicity tests, the green algae Pseudokirchneriella subcapitata was more sensitive to TiO₂ (IC25 1–2 mg/l) than C. dubia (IC25 9.4–26.4 mg/l) and the fathead minnow (IC25 values over 340 mg/l). Study results indicate that the specific organisms exposed and the effects of water quality parameters on TiO₂ toxicity should be considered in hazard evaluations of this nanoparticle.     

ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells

Titanium dioxide nanoparticles (TiO₂ NPs) are among the top five NPs used in consumer products, paints and pharmaceutical preparations. Since, exposure to such nanoparticles is mainly through the skin and inhalation, the present study was conducted in the human epidermal cells (A431). A mild cytotoxic response of TiO₂ NPs was observed as evident by the MTT and NR uptake assays after 48 h of exposure. However, a statistically significant (p < 0.05) induction in the DNA damage was observed by the Fpg-modified Comet assay in cells exposed to 0.8 μg/ml TiO₂ NPs (2.20 ± 0.26 vs. control 1.24 ± 0.04) and higher concentrations for 6 h. A significant (p < 0.05) induction in micronucleus formation was also observed at the above concentration (14.67 ± 1.20 vs. control 9.33 ± 1.00). TiO₂ NPs elicited a significant (p < 0.05) reduction in glutathione (15.76%) with a concomitant increase in lipid hydroperoxide (60.51%; p < 0.05) and reactive oxygen species (ROS) generation (49.2%; p < 0.05) after 6 h exposure. Our data demonstrate that TiO₂ NPs have a mild cytotoxic potential. However, they induce ROS and oxidative stress leading to oxidative DNA damage and micronucleus formation, a probable mechanism of genotoxicity. This is perhaps the first study on human skin cells demonstrating the cytotoxic and genotoxic potential of TiO₂ NPs.     

TiO2 nanoparticles induce oxidative DNA damage and apoptosis in human liver cells

Abstract

Titanium dioxide nanoparticles (TiO₂ NPs), widely used in consumer products, paints, pharmaceutical preparations and so on, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses in vitro and in vivo. The present study revealed that TiO₂ NPs induce significant (p < 0.05) oxidative DNA damage by the Fpg-Comet assay even at 1 µg/ml concentration. A corresponding increase in the micronucleus frequency was also observed. This could be attributed to the reduced glutathione levels with concomitant increase in lipid peroxidation and reactive oxygen species generation. Furthermore, immunoblot analysis revealed an increased expression of p53, BAX, Cyto-c, Apaf-1, caspase-9 and caspase-3 and decreased the level of Bcl-2 thereby indicating that apoptosis induced by TiO₂ NPs occurs via the caspase-dependent pathway. This study systematically shows that TiO₂ NPs induce DNA damage and cause apoptosis in HepG2 cells even at very low concentrations. Hence the use of such nanoparticles should be carefully monitored.     

DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells

Abstract

We investigated the genotoxic responses to two types of TiO₂ nanoparticles (<25 nm anatase: TiO₂-An, and <100 nm rutile: TiO₂-Ru) in human hepatoma HepG2 cells. Under the applied exposure conditions the particles were agglomerated or aggregated with the size of agglomerates and aggregates in the micrometer range, and were not cytotoxic. TiO₂-An, but not TiO₂-Ru, caused a persistent increase in DNA strand breaks (comet assay) and oxidized purines (Fpg-comet). TiO₂-An was a stronger inducer of intracellular reactive oxygen species (ROS) than TiO₂-Ru. Both types of TiO₂ nanoparticles transiently upregulated mRNA expression of p53 and its downstream regulated DNA damage responsive genes (mdm2, gadd45α, p21), providing additional evidence that TiO₂ nanoparticles are genotoxic. The observed differences in responses of HepG2 cells to exposure to anatase and rutile TiO₂ nanoparticles support the evidence that the toxic potential of TiO₂ nanoparticles varies not only with particle size but also with crystalline structure.     

Antigenotoxic potential of boron nitride nanotubes

Abstract

Boron and boron nitride nanotubes (BNNTs) are increasingly used in different industrial fields and, potentially, in some biomedical areas. As occurs with other nanomaterials (NMs), to increase our knowledge on their potential health hazards is a priority. Although in vitro approaches are a routine in getting biological information on the biological effects of NMs, the use of simple in vivo model organisms is receiving an increased interest. In this context, Drosophila melanogaster is widely used as a eukaryotic model for the study of the potential harmful effects associated with various agents, including NMs. The aim of this study is to provide new data on the potential antioxidant/antigenotoxic properties of boron and boron nitride nanotubes (BNNTs), as well as on other biological end-points. Our results show changes in the expression of genes involved in the antioxidant defense (CAT and SOD), and in those rel0061ted to the integrity of the intestinal barrier (Duox, Hml, Muc68D, and PPO2), at the highest exposure doses (5, 10?mM). However, non-relevant toxic or genotoxic effects were observed. Interestingly, BNNTs and boron significantly reduced the genotoxic effect of potassium dichromate (PDC), and the intracellular levels of reactive oxygen species (ROS). This suggest that the observed effects can be linked to the antioxidant properties of BNNTs and boron. This is the first study reporting antigenotoxicity/genotoxicity, and gene expression data, in the somatic cells of D. melanogaster larvae for BNNTs.     

Titanium dioxide nanoparticles prime a specific activation state of macrophages

Titanium dioxide nanoparticles (TiO₂ NPs) are widely used in foods, cosmetics, and medicine. Although the inhalation toxicity of TiO₂ NPs has been studied, the potential adverse effects of oral exposure of low-dose TiO₂ NPs are largely unclear. Herein, with macrophage cell lines, primary cells, and mouse models, we show that TiO₂ NPs prime macrophages into a specific activation state characterized by excessive inflammation and suppressed innate immune function. After a month of dietary exposure in mice or exposure in vitro to TiO₂ NPs (10 and 50?nm), the expressions of pro-inflammatory genes in macrophages were increased, and the expressions of anti-inflammatory genes were decreased. In addition, for macrophages exposed to TiO₂ NPs in vitro and in vivo, their chemotactic, phagocytic, and bactericidal activities were lower. This imbalance in the immune system could enhance the susceptibility to infections. In mice, after a month of dietary exposure to low doses of TiO₂ NPs, an aggravated septic shock occurred in response to lipopolysaccharide challenge, leading to elevated levels of inflammatory cytokines in serum and reduced overall survival. Moreover, TLR4-deficient mice and primary macrophages, or TLR4-independent stimuli, showed less response to TiO₂ NPs. These results demonstrate that TiO₂ NPs induce an abnormal state of macrophages characterized by excessive inflammation and suppressed innate immune function in a TLR4-dependent manner, which may suggest a potential health risk, particularly for those with additional complications, such as bacterial infections.     

Tissue distribution and excretion of intravenously administered titanium dioxide nanoparticles

As the biosafety of nanotechnology becomes a growing concern, the in vivo nanotoxicity of nanoparticles (NPs) has been drawn an increasing attention. Titanium dioxide nanoparticles (TiO₂-NPs) have been developed for versatile use, but the pharmacokinetics of intravenously administered TiO₂-NPs have not been investigated extensively. In the present study, the rutile-type TiO₂-NPs with a size about 20 nm were labeled with CF680 and ¹²⁵I. The labeled TiO₂-NPs were injected in mice or rats with the concentration of 1 mg/ml and the dose of 10 mg/kg body weight and their tissue distribution and excretion were investigated by using ex vivo fluorescent imaging, γ-counter and TEM. The results indicated that the TiO₂-NPs mainly accumulated in liver and spleen and could be retained for over 30 days in these tissues due to the phagocytosis by macrophages. The excretion assay found that the excretory rate of TiO₂-NPs through urine was higher than that of feces, indicating that renal excretion was the main excretion pathway of TiO₂-NPs. Overall results of the present study provided important information on distribution and excretion of TiO₂-NPs in vivo, which would greatly promote the pharmacokinetics and in vivo nanotoxicity research of TiO₂-NPs.     

Dissolution and bandgap paradigms for predicting the toxicity of metal oxide nanoparticles in the marine environment: an in vivo study with oyster embryos

Dissolution and bandgap paradigms have been proposed for predicting the ability of metal oxide nanoparticles (NPs) to induce oxidative stress in different in vitro and in vivo models. Here, we addressed the effectiveness of these paradigms in vivo and under conditions typical of the marine environment, a final sink for many NPs released through aquatic systems. We used ZnO and MnO₂ NPs as models for dissolution and bandgap paradigms, respectively, and CeO₂ NPs to assess reactive oxygen radical (ROS) production via Fenton-like reactions in vivo. Oyster embryos were exposed to 0.5–500?μM of each test NP over 24?h and oxidative stress was determined as a primary toxicity pathway across successive levels of biological complexity, with arrested development as the main pathological outcome. NPs were actively ingested by oyster larvae and entered cells. Dissolution was a viable paradigm for predicting the toxicity of NPs in the marine environment, whereas the surface reactivity based paradigms (i.e. bandgap and ROS generation via Fenton-like reaction) were not supported under seawater conditions. Bio-imaging identified potential cellular storage-disposal sites of solid particles that could ameliorate the toxicological behavior of non-dissolving NPs, whilst abiotic screening of surface reactivity suggested that the adsorption-complexation of surface active sites by seawater ions could provide a valuable hypothesis to explain the quenching of the intrinsic oxidation potential of MnO₂ NPs in seawater.     

Oxidative stress studies of six TiO2 and two CeO2 nanomaterials: Immuno-spin trapping results with DNA

Abstract

Six TiO₂ and two CeO₂ nanomaterials with dry sizes ranging from 6–410 nm were tested for their ability to cause DNA centered free radicals in vitro in the concentration range of 10–3,000 ug/ml. All eight of the nanomaterials significantly increased the adduction of the spin trap agent 5,5-dimethyl-1-pyroline N-oxide (DMPO) to DNA as measured by the experimental technique of immuno-spin trapping. The eight nanomaterials differed considerably in their potency, slope, and active concentration. The largest increase in DNA nitrone adducts was caused by a TiO₂ nanomaterial (25 nm, anatase) from Alfa Aesar. Some nanomaterials that increased the amount of DNA nitrone adducts at the lowest exposure concentrations (100 ug/ml) were Degussa TiO₂ (31 nm), Alfa Aesar TiO₂ (25 nm, anatase) and Nanoamor CeO₂ (8 nm, cerianite). At exposure concentrations of 10 or 30 ug/ml, no nanomaterials showed significant in vitro formation of DNA nitrone adducts.     

Endothelial cell activation,oxidative stress and inflammation induced by a panel of metal-based nanomaterials

Abstract

The importance of composition, size, crystal structure, charge and coating of metal-based nanomaterials (NMs) were evaluated in human umbilical vein endothelial cells (HUVECs) and/or THP-1 monocytic cells. Biomarkers of oxidative stress and inflammation were assessed because they are important in the development of cardiovascular diseases. The NMs used were five TiO₂ NMs with different charge, size and crystal structure, coated and uncoated ZnO NMs and Ag which were tested in a wide concentration range. There were major differences between the types of NMs; exposure to ZnO and Ag resulted in cytotoxicity and increased gene expression levels of HMOX1 and IL8. The intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) expression were highest in TiO₂ NM-exposed cells. There was increased adhesion of THP-1 monocytic cells onto HUVECs with Ag exposure. None of the NMs increased the intracellular ROS production. There were no major effects of the coating of ZnO NMs. The TiO₂ NMs data on ICAM-1 and VCAM-1 expression suggested that the anatase form was more potent than the rutile form. In addition, the larger TiO₂ NM was more potent than the smaller for gene expression and ICAM-1 and VCAM-1 expression. The toxicological profile of cardiovascular disease-relevant biomarkers depended on composition, size and crystal structure of TiO₂ NMs, whereas the charge on TiO₂ NMs and the coating of ZnO NMs were not associated with differences in toxicological profile.     

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.     

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO₂ NPs were used to compare the effects. Exposure to 32?μg/mL ZnO NPs (p?2 NPs (p?>?0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO₄ induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO₂ NP exposure (p?>?0.05). The presence of 250?nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p?p?>?0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p?>?0.05) except neutral red uptake assay (p?相似文献   

Continuous in vitro exposure of intestinal epithelial cells to E171 food additive causes oxidative stress,inducing oxidation of DNA bases but no endoplasmic reticulum stress

The whitening and opacifying properties of titanium dioxide (TiO₂) are commonly exploited when it is used as a food additive (E171). However, the safety of this additive can be questioned as TiO₂ nanoparticles (TiO₂-NPs) have been classed at potentially toxic. This study aimed to shed some light on the mechanisms behind the potential toxicity of E171 on epithelial intestinal cells, using two in vitro models: (i) a monoculture of differentiated Caco-2 cells and (ii) a coculture of Caco-2 with HT29-MTX mucus-secreting cells. Cells were exposed to E171 and two different types of TiO₂-NPs, either acutely (6–48?h) or repeatedly (three times a week for 3 weeks). Our results confirm that E171 damaged these cells, and that the main mechanism of toxicity was oxidation effects. Responses of the two models to E171 were similar, with a moderate, but significant, accumulation of reactive oxygen species, and concomitant downregulation of the expression of the antioxidant enzymes catalase, superoxide dismutase and glutathione reductase. Oxidative damage to DNA was detected in exposed cells, proving that E171 effectively induces oxidative stress; however, no endoplasmic reticulum stress was detected. E171 effects were less intense after acute exposure compared to repeated exposure, which correlated with higher Ti accumulation. The effects were also more intense in cells exposed to E171 than in cells exposed to TiO₂-NPs. Taken together, these data show that E171 induces only moderate toxicity in epithelial intestinal cells, via oxidation.     

Photocatalytic and phototoxic properties of TiO2-based nanofilaments: ESR and AFM assays

Abstract

There is uncertainty in understanding of the relationship between physico-chemical parameters of nanosized titanium dioxide (nano-TiO₂) and its toxicity when brought into contact with living cells. This study provides a multidisciplinary experimental insight into the toxicity and phototoxicity of the custom-made TiO₂-based nanowires (TiO₂-NWs). We employed electron spin resonance (ESR) to detect reactive oxygen species (ROS) generated in aqueous suspensions of TiO₂-NWs and combined these results with atomic force microscopy (AFM) to trace the onset of toxic effects towards human melanoma cells. The cells were treated with low concentrations (～2.5 μg/ml) of TiO₂-NWs and Degussa P25. High-resolution AFM surface topography and cell elasticity measurements revealed toxic effects both in cells incubated with TiO₂-NWs in the dark and exposed to the photo-oxidative stress under UVA radiation. In contrast to ROS generation efficacy in the absence of cells in vitro, no direct correlation was found between the physical parameters of nano-TiO₂ and cell toxicity.     

Oxidative stress increased hepatotoxicity induced by nano‐titanium dioxide in BRL‐3A cells and Sprague–Dawley rats

Extensive studies have shown that titanium dioxide (TiO₂) nanomaterials (NMs) can cause toxicity in vitro and in vivo under normal conditions. However, an adverse effect induced by nano‐TiO₂ in many diseased conditions, typically characterized by oxidative stress (OS), remains unknown. We investigated the toxicity of nano‐TiO₂ in rat liver cells (BRL‐3A) and Sprague–Dawley (SD) rat livers under OS conditions, which were generated using hydrogen peroxide (H₂O₂) in vitro and alloxan in vivo, respectively. In vitro results showed that cell death ratios after nano‐TiO₂ exposure were significantly enhanced (up to 2.62‐fold) in BRL‐3A cells under OS conditions, compared with normal controls. Significant interactions between OS conditions and nano‐TiO₂ resulted in the rapid G0/G1 to S phase transition and G2/M arrest, which were opposite to G0/G1 phase arrest in cells after NMs exposure only. In vivo results showed that obvious pathological changes in rat livers and the increased activities of four enzymes (i.e. aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and alkaline phosphatase) owing to liver damage after nano‐TiO₂ exposure under OS conditions, compared with their healthy controls. In addition, compared with increased hepatotoxicity after nano‐TiO₂ exposure, micro‐TiO₂ showed no adverse effects to cells and rat livers under OS conditions. Our results suggested that OS conditions synergistically increase nano‐TiO₂ induced toxicity in vitro and in vivo, indicating that the evaluation of nanotoxicity under OS conditions is essentially needed prior to various applications of NMs in foods, cosmetics and potential treatment of diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Modifications of the bacterial reverse mutation test reveals mutagenicity of TiO2 nanoparticles and byproducts from a sunscreen TiO2-based nanocomposite

The bacterial reverse mutation test, recommended by the Organization for Economic Co-operation and Development (OECD) to determine genotoxicity of chemical compounds, has been recently used by several authors to investigate nanoparticles. Surprisingly, test results have been negative, whereas in vitro mammalian cell tests often give positive genotoxic responses. In the present study, we used the fluctuation test procedure with the Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 to determine the mutagenic potential of TiO₂ nanoparticles (NP-TiO₂) and showed that, when it is used conventionally, this test is not suitable for nanoparticle genotoxicity assessment. Indeed, the medium used during exposure prevents electrostatic interactions between bacterial cells and nanoparticles, leading to false-negative responses. We showed that a simple pre-exposure of bacteria to NP-TiO₂ in a low ionic strength solution (NaCl 10 mM) at a pH below the nanoparticle isoelectric points (pH 5.5) can strongly improve the accuracy of the test. Thus, based on these improvements, we have demonstrated the genotoxicity of the engineered NP-TiO₂ tested and a NP-TiO₂ byproduct from a sunscreen nanocomposite. It was also shown that strain TA102 is more sensitive than the other strains, suggesting an oxidative stress-mediated mechanism of genotoxicity.     

Genotoxicity of phthalates

Many of the environmental, occupational and industrial chemicals are able to generate reactive oxygen species (ROS) and cause oxidative stress. ROS may lead to genotoxicity, which is suggested to contribute to the pathophysiology of many human diseases, including inflammatory diseases and cancer. Phthalates are ubiquitous environmental chemicals and are well-known peroxisome proliferators (PPs) and endocrine disruptors. Several in vivo and in vitro studies have been conducted concerning the carcinogenic and mutagenic effects of phthalates. Di(2-ethylhexyl)-phthalate (DEHP) and several other phthalates are shown to be hepatocarcinogenic in rodents. The underlying factor in the hepatocarcinogenesis is suggested to be their ability to generate ROS and cause genotoxicity. Several methods, including chromosomal aberration test, Ames test, micronucleus assay and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation test and Comet assay, have been used to determine genotoxic properties of phthalates. Comet assay has been an important tool in the measurement of the genotoxic potential of many chemicals, including phthalates. In this review, we will mainly focus on the studies, which were conducted on the DNA damage caused by different phthalate esters and protection studies against the genotoxicity of these chemicals.     

Toxicology of ZnO and TiO2 nanoparticles on hepatocytes: Impact on metabolism and bioenergetics

Abstract

Background and aim: Zinc oxide (ZnO) and titanium dioxide (TiO₂) nanomaterials (NMs) are used in many consumer products, including foodstuffs. Ingested and inhaled NM can reach the liver. Whilst their effects on inflammation, cytotoxicity, genotoxicity and mitochondrial function have been explored, no work has been reported on their impact on liver intermediary metabolism. Our aim was to assess the effects of sub-lethal doses of these materials on hepatocyte intermediary metabolism.

Material and methods: After characterisation, ZnO and TiO₂ NM were used to treat C3A cells for 4 hours at concentrations ranging between 0 and 10?μg/cm², well below their EC₅₀, before the assessment of (i) glucose production and glycolysis from endogenous glycogen and (ii) gluconeogenesis and glycolysis from lactate and pyruvate (LP). Mitochondrial membrane potential was assessed using JC-10 after 0–40?μg/cm² ZnO. qRT-PCR was used to assess phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression. Dihydroethidium (DHE) staining and FACS were used to assess intracellular reactive oxygen species (ROS) concentration.

Results: Treatment of cells with ZnO, but not TiO₂, depressed mitochondrial membrane potential, leading to a dose-dependent increase in glycogen breakdown by up to 430%, with an increase of both glycolysis and glucose release. Interestingly, gluconeogenesis from LP was also increased, up to 10-fold and correlated with a 420% increase in the PEPCK mRNA expression, the enzyme controlling gluconeogenesis from LP. An intracellular increase of ROS production after ZnO treatment could explain these effects.

Conclusion: At sub-lethal concentrations, ZnO nanoparticles dramatically increased both gluconeogenesis and glycogenolysis, which warrants further in vivo studies.