Titanium dioxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in human amnion epithelial (WISH) cells

Abstract

Titanium dioxide nanoparticles (TiO₂-NPs) induced cytotoxicity and DNA damage have been investigated using human amnion epithelial (WISH) cells, as an in vitro model for nanotoxicity assessment. Crystalline, polyhedral rutile TiO₂-NPs were synthesized and characterized using X-ray diffraction (XRD), UV–Visible spectroscopy, Fourier transform infra red (FTIR) spectroscopy, and transmission electron microscopic (TEM) analyses. The neutral red uptake (NRU) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assays revealed the concentration dependent cytotoxic effects of TiO₂-NPs (30.6 nm) in concentration range of 0.625–10 μg/ml. Cells exposed to TiO₂-NPs (10 μg/ml) exhibited significant reduction (46.3% and 34.6%; p < 0.05) in catalase activity and glutathione (GSH) level, respectively. Treated cells showed 1.87-fold increase in intracellular reactive oxygen species (ROS) generation and 7.3% (p < 0.01) increase in G₂/M cell cycle arrest, as compared to the untreated control. TiO₂-NPs treated cells also demonstrated the formation of DNA double strand breaks with 14.6-fold (p < 0.05) increase in Olive tail moment (OTM) value at 20 μg/ml concentration, vis-à-vis untreated control, under neutral comet assay conditions. Thus, the reduction in cell viability, morphological alterations, compromised antioxidant system, intracellular ROS production, and significant DNA damage in TiO₂-NPs exposed cells signify the potential of these NPs to induce cyto- and genotoxicity in cultured WISH cells.

Introduction

Titanium dioxide (TiO₂) is a naturally occurring mineral used in domestic and cosmetic products including anti-fouling paints, coatings, ceramics, additives in pharmaceuticals, food colorants (Jin et al., 2008, Vamanu et al., 2008), and as a sunscreen additive owing to its typical characteristics of surface adsorption, photo-catalysis and UV absorption (Douglas et al., 2000). Titanium either pure or in alloys is also extensively used for a wide range of implanted medical devices, such as dental implants, joint replacements, cardio-vascular stents, and spinal fixation devices. However, under mechanical stress or altered physiological conditions such as low pH, titanium-based implants can release large amounts of particle debris (4.47 mg/g dry tissue weight from titanium-alloy (Ti–6Al–4V) implants) both in the micrometer and nanometer size range (Brien et al., 1992, Buly et al., 1992, Arys et al., 1998, Cunningham et al., 2002). It is reported that the biological responses to nanoparticles (NPs) may exceed those elicited by micron-sized particles (Borm et al., 2006, Nel et al., 2006) due to their small size, high number per given mass, large specific surface area, and generation of free radicals (Lynch et al., 2006). The dimensions of the TiO₂-NPs are critical from the toxicity point of view, as the ultrafine TiO₂ causes more pronounced toxicity compared with fine TiO₂ particles (Driscoll and Maurer, 1991, Oberdörster et al., 1994, Oberdörster, 2000). Ultrafine TiO₂ particles (⩽20 nm) have been shown to induce impairment of macrophage function, persistently high inflammatory reactions, and increased pulmonary retention compared to fine TiO₂ (particle size > 200 nm) (Baggs et al., 1997). The TiO₂-NPs could be absorbed through inhalation, ingestion and dermal penetration into the body, and distributed in the important organs such as lung (Warheit et al., 2007, Wang et al., 2007), lymph nodes (Bermudez et al., 2004), brain (Thomas et al., 2006), liver and kidney (Wang et al., 2007).

There are growing concerns about the possible influence of NPs on human health, particularly with the exposures during prenatal, pregnancy or early childhood (Lacasana et al., 2005). Nanosized materials including the carboxylic polystyrene, gold and TiO₂-NPs are reported to cross the placental tissue (Semmler-Behnke et al., 2007, Tian et al., 2009). In an ex-vivo human placental perfusion model, Wick et al. (2010) demonstrated the uptake of nanosized fluorescently labeled polystyrene beads of 50, 80, 240, and 500 nm across the placental barrier. Also, in animal models, the translocation of TiO₂-NPs has been reported in brain of prenatally exposed mice. Since the blood barriers are under developed in the foetus, the NPs could easily pass into brain during the early stages of foetal development. The TiO₂-NPs in anatase (crystals of eight-faced tetragonal dipyramids) form, administered subcutaneously to pregnant ICR mice, were found to be transferred to and affected the genital and cranial nerve systems of the offspring (Takeda et al., 2009).

Furthermore, Gurr et al. (2005) demonstrated that anatase-sized (10 and 20 nm) TiO₂-NPs in the absence of photoactivation induce oxidative DNA damage, lipid peroxidation, and micronuclei formation, and cause increased hydrogen peroxide and nitric oxide production in human bronchial epithelial (BEAS-2B) cell line. Vevers and Jha (2008) have reported the enhanced level of TiO₂-NPs induced DNA damage in presence of UV light in rainbow trout gonadal tissue cells. Whereas, in the goldfish skin cells (GFSk-S1), the TiO₂-NPs caused DNA damage in the absence of UV light (Reeves et al., 2008). Also, in human monoblastoid and bronchial epithelial cells, the TiO₂-NPs induce apoptosis mainly by destabilizing the lysosomal membrane and lipid peroxidation (Vamanu et al., 2008, Zhao et al., 2009, Hussain et al., 2010). The TiO₂ induced DNA damage and apoptosis have also been demonstrated in human lymphocytes, U937 human monoblastoid cells, A549 alveolar epithelial cells, NRK-52E normal rat kidney cells, and A431 human epidermal cells (Vamanu et al., 2008, Gopalan et al., 2009, Park et al., 2007, Barillet et al., 2010, Shukla et al., 2011). However, to the best of our understanding, no systematic study on assessment of the TiO₂-NPs cytotoxicity and genotoxicity on cells of placental origin are reported in literature. There are reports that the human amnion epithelial cell line established as WISH (Wistar Institute, Susan Hayflick) cells maintains the similar characteristics of growth, cell morphology, prostaglandin production, and susceptibility to apoptotic agents, as the primary amnion cells (Lundgren et al., 1997, Moore et al., 2002). Perhaps, the stability of these cells makes them more useful for studies that the primary cultures of amnion cells will not tolerate (Kumar et al., 2004). Therefore, the amniotic WISH cells have been chosen as a model in this study, with the aim to assess the effects of sonomechanically synthesized TiO₂-NPs on the (i) cell viability as cytotoxic end point, (ii) intracellular ROS production and antioxidative enzymes, (iii) induction of DNA strand breaks, and (iv) progression of normal cell cycle, in order to elucidate the plausible role of TiO₂-NPs in evoking toxic responses in WISH cells, as putative markers for placental toxicity.