Investigation of the in vitro genotoxicity of two rutile TiO2 nanomaterials in human intestinal and hepatic cells and evaluation of their interference with toxicity assays

Abstract

TiO 2 nanomaterials (NM) have a wide range of industrial applications, including their use in food products. The incorporation of these NMs in consumer products represents a clear concern for public health safety agencies and consumers, and further investigation of the potential impact of these products on human health is necessary. Indeed, since human oral exposure to TiO 2 NMs is expected to increase in the years to come, there exist legitimate concerns about the risk assessment of these nanomaterials present in food products. A considerable amount of studies investigating the adverse effects of TiO 2 NMs have focused on the genotoxic effects of these NMs, and more recently they have been classified by the International Agency for Research on Cancer (IARC) as carcinogen group 2B following inhalation studies (IARC 2010). While numerous data are available for anatase or mixes of anatase/rutile forms, the toxicity and the genotoxicity of rutile TiO 2 NMs have been rarely investigated. The aim of our study was therefore to investigate the cytotoxic and genotoxic effects of two rutile TiO 2 NMs, differing in surface coating, NM103 (hydrophobic) and NM104 (hydrophilic), on intestinal and hepatic cell models. Following 3 or 24 h treatments with concentrations of TiO 2 NMs from 1.2 to 80 µg/cm 2 , we have assessed the genotoxicity of these NMs with H2AX, alkaline comet assay and micronucleus (MN) assays. Cellular viability and effects on oxidative stress were also evaluated. Although TEM imaging demonstrated the presence of the two TiO 2 NMs within the cytoplasm, no significant cytotoxic or genotoxic were observed in either cell model. We have also evaluated and taken into account a variety of potential sources of interference of NMs with cellular assays. TiO 2 NMs present in the cytoplasm introduce uncertainty in the scoring of micronuclei, and therefore this assay is not recommended for the evaluation of the genotoxicity of TiO 2 NMs, or other NMs demonstrating similar interference. The unique properties of TiO 2 NMs introduce additional complexity for genotoxicity testing, and caution must be taken in order to obtain reliable results necessary for accurate hazard assessment.

Simultaneous Quantification and Visualization of Titanium Dioxide Nanomaterial Uptake at the Single Cell Level in an In Vitro Model of the Human Small Intestine

“…It was suggested that the transport mechanisms were disturbed by TiO 2 NM ingestion . This goes along with our findings, where decreased zinc and iron cellular concentrations were observed, even under nontoxic exposure conditions . Furthermore, the analysis of protein corona of TiO 2 NMs revealed different amounts of metal binding proteins (albumin, iron export ABC transporter ATPase, ovotransferrin, and tetranectin) and membrane transport proteins (solute carrier family) associated with NM surface (S 1).…”

“…Visualization, internalization, and quantification of TiO 2 NMs were studied at a single cell level by means of IBM, ToF‐SIMS, and TEM techniques. Caco‐2 cells were exposed to the TiO 2 NMs at nontoxic conditions . Two different concentrations (20 and 100 µg mL −1 , corresponding to 4.7 and 24.4 µg cm −2 ) of hydrophobic NM103 or hydrophilic NM104 TiO 2 at two different time points (12 and 24 h) were chosen for IBM visualization and quantification.…”

Analysis of the Characteristics and Cytotoxicity of Titanium Dioxide Nanomaterials Following Simulated In Vitro Digestion

“…The absence of cytotoxicity of the TiO 2 in Caco-2 cells is in agreement with previous reports [24,25,36,37]. Although Gerloff et al [36] and Dorier et al [24,37] did not use exactly the same benchmark NMs, they used TiO 2 very similar to ours.…”

“…In spite of many papers reporting the cytotoxicity assessment of TiO 2 in cells from GIT (see for example [24,25]), none has addressed and compared the effects upon digestion process of different crystalline TiO 2 forms in two distinct cell lines. A recent work studied the dissolution behavior, biodurability and persistence of several NMs (TiO 2 , SiO 2 , ZnO and Fe 2 O 3 ) in individual simulated gastrointestinal fluids (saliva, gastric and intestinal) and a physiologically relevant digestion cascade (saliva-gastric-intestinal), showing that TiO 2 was the most biodurable and persistent NM [26].…”

Genotoxic Impact of Aluminum-containing Nanomaterials in Human Intestinal and Hepatic Cells.

“…The human colorectal adenocarcinoma Caco-2 cell line was cultured (passages 25-38) until differentiation after 21 days as described in [40] including for cell seeding in various plate formats depending on the assay performed. Simarly, HepaRG cells (passages 13-19) were cultured and seeded for the various assays as previously described [40,41]. 10% FBS or William's medium + 5% FBS respectively.…”