Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents

Titanium dioxide nanoparticles (TiO₂-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1–50?μg/mL TiO₂-NPs, 86% anatase/14% rutile, 24?±?6?nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO₂-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO₂-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO₂-NPs.     

The in vivo underlying mechanism for recovery response formation in nano-titanium dioxide exposed Caenorhabditis elegans after transfer to the normal condition

So far, we still know little about mechanism for recovery response of engineered nanomaterials (ENMs). Here we used Caenorhabditis elegans to investigate recovery responses of titanium dioxide nanoparticles (TiO₂-NPs) exposed animals and the underlying mechanism. After acute exposure to TiO₂-NPs (100 mg/L), endpoints including defecation and permeable state of intestinal barrier of exposed nematodes returned to control levels; however, after prolonged exposure to TiO₂-NPs (100 μg/L), endpoints of exposed nematodes could not be recovered to control levels under the normal condition. After prolonged exposure to TiO₂-NPs, nematodes exhibited severe deficits in development of intestinal barrier and AVL and DVB neurons controlling defecation; however, after acute exposure to TiO₂-NPs, nematodes had normal developmental state of intestinal barrier and AVL and DVB neurons. Our results imply that developmental states of intestinal barrier and AVL and DVB neurons may serve as a pivotal determinant for recovery response in TiO₂-NPs exposed nematodes.From the Clinical EditorThis basic science study investigates the recovery response to TiO₂ nanoparticles in a nematode model, and concludes that developmental states of the intestinal barrier and AVL and DVB neurons likely serve as determinants for recovery following TiO2-NP exposure.     

Comparison of manganese oxide nanoparticles and manganese sulfate with regard to oxidative stress, uptake and apoptosis in alveolar epithelial cells

Due to their physicochemical characteristics, metal oxide nanoparticles (NPs) interact differently with cells compared to larger particles or soluble metals. Oxidative stress and cellular metal uptake were quantified in rat type II alveolar epithelial cells in culture exposed to three different NPs: manganese(II,III) oxide nanoparticles (Mn₃O₄-NPs), the soluble manganese sulfate (Mn-salt) at corresponding equivalent doses, titanium dioxide (TiO₂-NPs) and cerium dioxide nanoparticles (CeO₂-NPs). In the presence of reactive oxygen species an increased apoptosis rate was hypothesized. Oxidative stress was assessed by detection of fluorescently labeled reactive oxygen species and by measuring intracellular oxidized glutathione. Catalytic activity was determined by measuring catalyst-dependent oxidation of thiols (DTT-assay) in a cell free environment. Inductively coupled plasma mass spectrometry was used to quantify cellular metal uptake. Apoptosis rate was determined assessing the activity of caspase-3 and by fluorescence microscopic quantification of apoptotic nuclei. Reactive oxygen species were mainly generated in cells treated with Mn₃O₄-NPs. Only Mn₃O₄-NPs oxidized intracellular glutathione. Catalytic activity could be exclusively shown for Mn₃O₄-NPs. Cellular metal uptake was similar for all particles, whereas Mn-salt could hardly be detected within the cell. Apoptosis was induced by both, Mn₃O₄-NPs and Mn-salt. The combination of catalytic activity and capability of passing the cell membrane contributes to the toxicity of Mn₃O₄-NPs. Apoptosis of samples treated with Mn-salt is triggered by different, potentially extracellular mechanisms.     

Susceptible genes regulate the adverse effects of TiO2-NPs at predicted environmental relevant concentrations on nematode Caenorhabditis elegans

Contributions from mutations of susceptible genes to TiO₂-NPs toxicity at environmental relevant concentrations (ERCs) and the underlying mechanism are largely unclear. After prolonged exposure, among the examined 19 mutants associated with oxidative stress or stress response, we show that sod-2, sod-3, mtl-2, and hsp-16.48 were susceptible genes for TiO₂-NPs toxicity on reproduction and locomotion behavior, sod-2, sod-3, and mtl-2 were susceptible genes for TiO₂-NPs toxicity on survival and intestinal development, and mtl-2 was susceptible gene for TiO₂-NPs toxicity on development. Mutations of these susceptible genes, together with sensitive endpoints, could be used to evaluate TiO₂-NPs toxicity at the concentration of 0.0001 μg/L. Our results imply the usefulness of identified susceptible genes in assessing the potential nanotoxicity of engineered nanomaterial (ENM) at ERCs. One important mechanism to explain property of identified susceptible genes for TiO₂-NPs toxicity was that mutations of these susceptible genes enhanced the uptake of TiO₂-NPs into body of nematodes.From the Clinical EditorThis team of authors identified susceptibility genes influencing the uptake and consequential toxicity of TiO₂ nanoparticles in a nematode, highlighting the general importance of investigating genetic influence on nanoparticle delivery.     

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.     

Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells

Despite that applications of titanium dioxide nanoparticles (TiO₂-NPs) have been developed in the fields of paints, waste water treatment, sterilization, cosmetics, food additive, bio-medical ceramic and implant biomaterials and so on, relatively few studies have been conducted to determine the neurotoxicity of TiO₂-NPs exposure. In the present study, we investigated the cytotoxicity of TiO₂-NPs using PC12 cells and intended to clarify the molecular mechanisms underlying the biological effects of TiO₂-NPs. PC12 cell is a type of cells, which have been used as an in vitro model of dopaminergic neurons for neurodegenerative diseases research. In addition, the roles of the particle size and crystal structure of TiO₂-NPs to the neurotoxicity were also investigated. The anatase TiO₂-NPs displayed a dose-dependent behavior on decreasing cell viability, increasing levels of lactate dehydrogenase (LDH), activating oxidative stress, inducing apoptosis, disturbing cell cycle, triggering JNK- and p53-mediated signaling pathway. In comparison to anatase TiO₂-NPs, the rutile TiO₂-NPs showed moderately toxic effect on neuron cells. The micron-sized TiO₂ did not exhibit any toxic response. It is suggested from our results that reactive oxygen species (ROS) have a mediation effect to oxidative stress and up-regulation of JNK and P53 phosphorylation involved in mechanistic pathways of TiO₂-NPs can induce apoptosis and cell cycle arrest in PC12 cells. In addition, both the size and crystal structure of TiO₂-NPs exposure contributed to the neurotoxicity. Nanoparticles were more toxic than micrometer-sized particles and the anatase form were more toxic than the rutile.     

Titanium dioxide nanoparticle exposure alters metabolic homeostasis in a cell culture model of the intestinal epithelium and Drosophila melanogaster

Nanosized titanium dioxide (TiO₂) is a common additive in food and cosmetic products. The goal of this study was to investigate if TiO₂ nanoparticles affect intestinal epithelial tissues, normal intestinal function, or metabolic homeostasis using in vitro and in vivo methods. An in vitro model of intestinal epithelial tissue was created by seeding co-cultures of Caco-2 and HT29-MTX cells on a Transwell permeable support. These experiments were repeated with monolayers that had been cultured with the beneficial commensal bacteria Lactobacillus rhamnosus GG (L. rhamnosus). Glucose uptake and transport in the presence of TiO₂ nanoparticles was assessed using fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). When the cell monolayers were exposed to physiologically relevant doses of TiO₂, a statistically significant reduction in glucose transport was observed. These differences in glucose absorption were eliminated in the presence of beneficial bacteria. The decrease in glucose absorption was caused by damage to intestinal microvilli, which decreased the surface area available for absorption. Damage to microvilli was ameliorated in the presence of L. rhamnosus. Complimentary studies in Drosophila melanogaster showed that TiO₂ ingestion resulted in decreased body size and glucose content. The results suggest that TiO₂ nanoparticles alter glucose transport across the intestinal epithelium, and that TiO₂ nanoparticle ingestion may have physiological consequences.     

Close association of intestinal autofluorescence with the formation of severe oxidative damage in intestine of nematodes chronically exposed to Al(2)O(3)-nanoparticle

In nematodes, acute exposure (24-h) to 8.1–30.6 mg/L Al₂O₃-nanoparticles (NPs) or Al₂O₃ did not influence intestinal autofluorescence, whereas chronic exposure (10-d) to Al₂O₃-NPs at concentrations of 8.1–30.6 mg/L or Al₂O₃ at concentrations of 23.1–30.6 mg/L induced significant increases of intestinal lipofuscin accumulation, and formation of severe stress response and oxidative damage in intestines. Moreover, significant differences of intestinal autofluorescence, stress response and oxidative damage in intestines of Al₂O₃-NPs exposed nematodes from those in Al₂O₃ exposed nematodes were detected at examined concentrations. Oxidative damage in intestine was significantly correlated with intestinal autofluorescence in exposed nematodes, and oxidative damage in intestine was more closely associated with intestinal autofluorescence in nematodes exposed to Al₂O₃-NPs than exposed to Al₂O₃. Thus, chronic exposure to Al₂O₃-NPs may cause adverse effects on intestinal lipofuscin accumulation by inducing the formation of more severe oxidative stress in intestines than exposure to Al₂O₃ in nematodes.     

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.     

Proteomic analyses of early response of unicellular eukaryotic microorganism Tetrahymena thermophila exposed to TiO2 particles

Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO₂ nanoparticles (TiO₂-NPs) and their bulk counterparts (bulk-TiO₂). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO₂-NPs or bulk-TiO₂ particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE?+?nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO₂ particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO₂-NPs concentration differed significantly from the response to higher TiO₂-NPs concentration and both bulk-TiO₂ concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO₂; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO₂-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.     

Intracellular distribution,geno- and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells

Nanomaterials are defined as substances with at least one dimension smaller than 100 nm in size and are used for a multitude of purposes. Titanium dioxide nanoparticles (TiO₂-NPs) are an important material used as an additive in pharmaceutical and cosmetic products. Due to their high surface-to-mass index, TiO₂ nanoparticles show different physical and chemical characteristics compared to the bulk substance. The knowledge about geno- or cytotoxic effects of TiO₂-NPs is incomplete since existing studies show contrary results.     

Effects of the chronic exposure to cerium dioxide nanoparticles in Oncorhynchus mykiss: Assessment of oxidative stress,neurotoxicity and histological alterations

Cerium dioxide nanoparticles (CeO₂-NPs) have a variety of uses, especially in the production of solar panels, oxygen pumps, gas sensors, computer chips and catalytic converters. Despite their worldwide use, the few published studies demonstrate that metallic nanoparticles, in general, are still not properly characterized in terms of their potencial ecotoxicological effects. CeO₂-NPs, in particular, have demonstrated extreme antioxidant activity, but their in vivo toxicity is still unknown. This work intended to characterize the chronic toxicity (28 days) of three different ecologically relevant concentrations (0.1, 0.01, and 0.001 μg/L) of CeO₂-NPs in the rainbow trout (Oncorhynchus mykiss), in terms of biomarkers of oxidative stress [activity of the enzymes glutathione S-transferases (GSTs) and catalase (CAT)] and neurotoxicity [activity of the enzyme acetylcholinesterase (AChE)], as well as histological alterations in liver and gills. In the hereby study, GSTs activity was increased in gills of fish exposed to the highest CeO₂–NPs level. Moreover, a potential anti-oxidant response was also reported, with a significant increase of CAT activity observed in livers of the same fish. AChE, however, was not significantly altered in fish eyes. Individuals exposed to CeO₂-NPs also presented marked changes in the gills (e.g. epithelial lifting, intercellular edema, lamellar hypertrophy and hyperplasia, secondary lamella fusion and aneurysms) and liver (e.g. hepatocyte vacuolization, pyknotic nucleus, enlargement of sinusoids and hyperemia). The semi-quantitative analysis (organs pathological index) also showed the establishment of a dose-effect relationship. Further studies about the ecotoxicological effects of the CeO₂-NPs have yet to be conducted, considering their properties, as the aggregation chemistry and the ratio of its redox state, which may affect their availability to the organism and their toxicity in the environment and biota.     

Toxicity of TiO2 nanoparticles on soil nitrification at environmentally relevant concentrations: Lack of classical dose–response relationships

Titanium-dioxide nanoparticles (TiO₂-NPs) are increasingly released in agricultural soils through, e.g. biosolids, irrigation or nanoagrochemicals. Soils are submitted to a wide range of concentrations of TiO₂-NPs depending on the type of exposure. However, most studies have assessed the effects of unrealistically high concentrations, and the dose–response relationships are not well characterized for soil microbial communities. Here, using soil microcosms, we assessed the impact of TiO₂-NPs at concentrations ranging from 0.05 to 500?mg kg^?1?dry-soil, on the activity and abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB), and nitrite-oxidizing bacteria (Nitrobacter and Nitrospira). In addition, aggregation and oxidative potential of TiO₂-NPs were measured in the spiking suspensions, as they can be important drivers of TiO₂-NPs toxicity. After 90?days of exposure, non-classical dose–response relationships were observed for nitrifier abundance or activity, making threshold concentrations impossible to compute. Indeed, AOA abundance was reduced by 40% by TiO₂-NPs whatever the concentration, while Nitrospira was never affected. Moreover, AOB and Nitrobacter abundances were decreased mainly at intermediate concentrations nitrification was reduced by 25% at the lowest (0.05?mg?kg^?1) and the highest (100 and 500?mg?kg^?1) TiO₂-NPs concentrations. Path analyses indicated that TiO₂-NPs affected nitrification through an effect on the specific activity of nitrifiers, in addition to indirect effects on nitrifier abundances. Altogether these results point out the need to include very low concentrations of NPs in soil toxicological studies, and the lack of relevance of classical dose–response tests and ecotoxicological dose metrics (EC50, IC50…) for TiO₂-NPs impact on soil microorganisms.     

A review of the fate of inhaled α-quartz in the lungs of rats

The distribution of dust particles within the lungs and their excretion are highly associated with their pulmonary toxicity. Literature was reviewed to discern pulmonary translocation pathways for inhaled α-quartz compared to those for inhaled TiO₂. Accordingly, it was hypothesized α-quartz particles in the alveoli were phagocytized by alveolar macrophages but silica-containing macrophages remained in the alveoli for longer time in contrast to the rapid elimination from the alveoli seen for TiO₂-containing macrophages. In addition, it was presumed that free silica particles are translocated in the interstitium, possibly through the cytoplasm of Type I epithelial cells, as observed with TiO₂. Free silica particles are presumed to be phagocytized by interstitial macrophages soon after the particles penetrate the interstitium; these dust cells are then translocated to the ciliated airway regions in the lumen through bronchus-associated lymphoid tissue (BALT). The pulmonary retention half-time of dust particles in rats exposed to α-quartz is several times longer than that of rats exposed to TiO₂, as long as the lung dust burden is ≈ 3?mg. The reduced pulmonary particle clearance ability in rats exposed to α-quartz aerosol is presumably attributed to the long-term retention of dust cells both in the alveoli and in the interstitium; this retention may be caused by the reduced chemotactic abilities of α-quartz-containing dust cells. However, the accumulation of α-quartz-containing dust cells in the lungs is not associated with the occurrence of pulmonary inflammation.     

TiO2 nanoparticles induce endothelial cell activation in a pneumocyte–endothelial co-culture model

The effects of particulate matter (PM) on endothelial cells have been evaluated in vitro by exposing isolated endothelial cells to different types of PM. Although some of the findings from these experiments have been corroborated by in vivo studies, an in vitro model that assesses the interaction among different cell types is necessary to achieve more realistic assays. We developed an in vitro model that mimics the alveolar–capillary interface, and we challenged the model using TiO₂ nanoparticles (TiO₂-NPs). Human umbilical endothelial cells (HUVECs) were cultured on the basolateral side of a membrane and pneumocytes (A549) on the apical side. Confluent co-cultures were exposed on the apical side to 10 μg/cm² of TiO₂-NPs or 10 ng/mL of TNFα for 24 h. Unexposed cultures were used as negative controls. We evaluated monocyte adhesion to HUVECs, adhesion molecule expression, nitric oxide concentration and proinflammatory cytokine release. The TiO₂-NPs added to the pneumocytes induced a 3- to 4-fold increase in monocyte adhesion to the HUVECs and significant increases in the expression of adhesion molecules (4-fold for P-selectin at 8 h, and about 8- and 10-fold for E-selectin, ICAM-1, VCAM-1 and PECAM-1 at 24 h). Nitric oxide production also increased significantly (2-fold). These results indicate that exposing pneumocytes to TiO₂-NPs causes endothelial cell activation.     

Comparison of the effects of MnO2-NPs and MnO2-MPs on mitochondrial complexes in different organs

Today, nanoparticles (NPs) have been widely used in various fields. Manganese oxide nanoparticles have attracted a lot of attention due to many applications. One of the major concerns regarding the widespread use of various NPs is the exposure and accumulation in human organs and finally toxicity. The generation of reactive oxygen species (ROS) by mitochondria is one of the most important mechanisms of toxicity suggested by published studies induced by other NPs. However, limited studies have been conducted on the mechanism of toxicity of MnO₂-NPs and MnO₂-microparticles (MnO₂-MPs). In this study, we compared the accumulation of MnO₂-NPs and MnO₂-MPs in different tissues and evaluated their effects on mitochondrial complexes in isolated mitochondria. Our results showed that intravascular (iv) administration of the MnO₂-NPs in the same dose compared to the MnO₂-MPs resulted in more accumulation in the C57 mouse female tissues. The effect of MnO₂-NPs and MnO₂-MPs in mitochondria showed that complexes I and III play an important role in increasing ROS generation and this effect is related to type of tissue. Also, our results showed that exposure to MnO₂-NPs and MnO₂-MPs reduced the activity of mitochondrial complexes II and IV. Our results suggest that the toxicity of the MnO₂-NPs is higher than that of the MnO₂-MPs and can lead to the depletion of antioxidant status, likely induction of apoptosis, cancer, and neurodegenerative disease.

Abbreviations: NPs: nanoparticles; ROS: reactive oxygen species; SDH: succinate dehydrogenase; DCFH-DA: dichloro-dihydro-fluorescein diacetate; ELISA: enzyme-linked immunosorbent assay; MnO2-NPs: manganese oxide nanoparticles     

Risk assessment of titanium dioxide nanoparticles via oral exposure,including toxicokinetic considerations

Titanium dioxide white pigment consists of particles of various sizes, from which a fraction is in the nano range (<100?nm). It is applied in food as additive E 171 as well as in other products, such as food supplements and toothpaste. Here, we assessed whether a human health risk can be expected from oral ingestion of these titanium dioxide nanoparticles (TiO₂ NPs), based on currently available information. Human health risks were assessed using two different approaches: Approach 1, based on intake, i.e. external doses, and Approach 2, based on internal organ concentrations using a kinetic model in order to account for accumulation over time (the preferred approach). Results showed that with Approach 1, a human health risk is not expected for effects in liver and spleen, but a human health risk cannot be excluded for effects on the ovaries. When based on organ concentrations by including the toxicokinetics of TiO₂ NPs (Approach 2), a potential risk for liver, ovaries and testes is found. This difference between the two approaches shows the importance of including toxicokinetic information. The currently estimated risk can be influenced by factors such as absorption, form of TiO₂, particle fraction, particle size and physico-chemical properties in relation to toxicity, among others. Analysis of actual particle concentrations in human organs, as well as organ concentrations and effects in liver and the reproductive system after chronic exposure to well-characterized TiO₂ (NPs) in animals are recommended to refine this assessment.     

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.