Nanotoxicity of TiO2 nanoparticles to erythrocyte in vitro
Introduction
The revolution in nanotechnology brings advantages in diverse areas of our lives such as engineering, information technology and medicine, etc. (Gross, 1999, Kim et al., 2005, Akerman et al., 2002). However, recent studies also had suggested that nano-materials with their tiny size (0.1–100 nm) could easily enter into human body (Kreyling et al., 2002, Takenaka et al., 2001). The small size and high surface-volume ratio endow them with an active group or intrinsic toxicity (Hoet et al., 2004; Donaldson and Tran, 2002, Warheit, 2004). Rationally the widespread application of nano-products would arise the concerns about the nano-thing risk on human being health because of their size and high reactive surface, totally differing from their bulk materials (Donaldson et al., 2000, Oberdörster et al., 2005, Dingman, 2008).
Titanium dioxide (TiO2) has been utilized for many years as an inert, non-toxic pigment product or other substance evaluated by many regulatory bodies such as the Material Safety Data Sheets (MSDS) and others charged with the responsibility of safeguarding the health of occupational workers and public health safety. The US Food and Drug Administration (FDA) allowed for its ingestion as drug additive, external application including the eye area, and considered it as a safe substance for public health (FDA, 2002). Based on these conclusions, many organizations and businesses have extrapolated the safety assessment to TiO2 nanoparticles (nano-TiO2), which actually have been used as sunscreen additive for years with three characteristics: surface adsorption, photo-catalysis and UV absorption (Douglas et al., 2000). However, several studies have challenged the permitted use of nano-TiO2 (Service, 2003, Warheit, 2004, Afaq et al., 1998, Warheit et al., 2007, Wang et al., 2007, Thrall, 2006). The importance is that many data, yet limited, suggest that nano-TiO2 could be absorbed through respiratory tract (inhalation), digestive tract (ingestion) and dermal (penetration), into body and distributed in such key organs as lung (Warheit et al., 2007, Wang et al., 2007), lymph nodes (Bermudez et al., 2004), liver (Wang et al., 2007, Jani et al., 1994), kidney (Wang et al., 2007) and brain (Thomas et al., 2006), etc. As we know, nano-TilO2 has to travel along the body within blood before arriving at target organs. So the influence of nano-TiO2 on cells in blood should be paid more attention to for safety application. Although playing a vital role in carrying oxygen from lungs to tissues or organs to meet metabolic needs, erythrocyte, dominant (99%) cell in the blood, is vulnerable to toxicity with deformation, agglutination and membrane damage (Kim et al., 2005, Rothen-Rutishauser et al., 2006). So it is necessary and urgent to assess the risk of nano-TiO2 on erythrocyte. Moreover, erythrocyte toxicity has also been a routine to carry out for new chemicals research (Prasanthi et al., 2005, Lee et al., 2004). Unfortunately, up to now, little is known about the interaction of the nano-TiO2 with erythrocyte (Nemmar et al., 2002).
In the present study, we systematically research effects of nano-TiO2 on erythrocyte. Firstly the influence of nano-TiO2 on erythrocyte sedimentation was detected. Secondly, hemagglutination induced by nano-TiO2 was studied by optical microscopy. Finally, as ghost cell was found in some cases, hemolysis was investigated under nano-TiO2 at different concentrations. Furthermore the mechanism of such adverse effects was studied by using ultra-thin cell section with transmission electron microscope (TEM) and by using oxidative stress theory with malondialdehyde (MDA) quantified in the culture fluid of erythrocyte under nano-TiO2.
Section snippets
Materials
Anatase nano-TiO2 was kindly provided by Su-ping Qian (Nanometer craft technical monopoly inventor from Shanghai branch of the Academy of Sciences of China). TiO2 microparticles (micro-TiO2, 99.8%, anatase, CAS No. 1317-70-0) were purchased from Sigma–Aldrich. Both TiO2 particles were diluted by phosphate buffered solution (PBS, pH 7.4) and sonicated for 5 min prior to incubation with erythrocytes in order to avoid aggregation and stored at 4 °C. MDA Detection Kit was purchased from Nanjing
Size of TiO2 observed by TEM
As shown in Fig. 1, nano-TiO2 was with the size about 20 nm (a). The size for micro-TiO2 was about 200 nm (b). The size was consistent with the data provided by producer.
Erythrocyte sedimentation and agglutination
Erythrocyte sedimentation rate (ESR) is to observe the height to which the erythrocytes fall in a given time interval, usually 1 h. In general, the sedimentation under gravity can monitor the dramatic abnormal alteration in shape and membrane of erythrocyte induced by diverse toxicants.
To our surprise, as shown in Fig. 2, the
Discussion
As we know, ultrafine particles in air are defined as particles in size less than 100 nm, compared with fine particles (>100 nm). The size falls into the category of nano-scale. Nanotoxicity is a new term and many data about nanotoxicity are firstly derived from epidemiological studies about ultrafine particles such as pneumoconiosis. In addition, generally accepted nomenclature of nanosphere includes nanoparticles, ultrafine particles and quantum dots.
Evidence from epidemiological studies
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 50572074 and 50673078), the Shanghai Key Fundamental Project (Grant No. 06JC14068) and the Innovation Program of Shanghai Municipal Education Commission (Grant No. 08ZZ21).
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