Elsevier

Toxicology in Vitro

Volume 19, Issue 7, October 2005, Pages 975-983
Toxicology in Vitro

In vitro toxicity of nanoparticles in BRL 3A rat liver cells

https://doi.org/10.1016/j.tiv.2005.06.034Get rights and content

Abstract

This study was undertaken to address the current deficient knowledge of cellular response to nanosized particle exposure. The study evaluated the acute toxic effects of metal/metal oxide nanoparticles proposed for future use in industrial production methods using the in vitro rat liver derived cell line (BRL 3A). Different sizes of nanoparticles such as silver (Ag; 15, 100 nm), molybdenum (MoO3; 30, 150 nm), aluminum (Al; 30, 103 nm), iron oxide (Fe3O4; 30, 47 nm), and titanium dioxide (TiO2; 40 nm) were evaluated for their potential toxicity. We also assessed the toxicity of relatively larger particles of cadmium oxide (CdO; 1 μm), manganese oxide (MnO2; 1–2 μm), and tungsten (W; 27 μm), to compare the cellular toxic responses with respect to the different sizes of nanoparticles with different core chemical compositions. For toxicity evaluations, cellular morphology, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH) levels, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were assessed under control and exposed conditions (24 h of exposure). Results showed that mitochondrial function decreased significantly in cells exposed to Ag nanoparticles at 5–50 μg/ml. However, Fe3O4, Al, MoO3 and TiO2 had no measurable effect at lower doses (10–50 μg/ml), while there was a significant effect at higher levels (100–250 μg/ml). LDH leakage significantly increased in cells exposed to Ag nanoparticles (10–50 μg/ml), while the other nanoparticles tested displayed LDH leakage only at higher doses (100–250 μg/ml). In summary the Ag was highly toxic whereas, MoO3 moderately toxic and Fe3O4, Al, MnO2 and W displayed less or no toxicity at the doses tested. The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, reduced mitochondrial membrane potential and increase in ROS levels, which suggested that cytotoxicity of Ag (15, 100 nm) in liver cells is likely to be mediated through oxidative stress.

Introduction

Nanotechnology involves the creation and manipulation of materials at nanoscale levels to create products that exhibit novel properties. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyballs) and quantum dots have received enormous attention to create new types of analytical tools for biotechnology and life sciences (Bruchez et al., 1998, Taton et al., 2000, Cui et al., 2001). Nanomaterials, which range in size from 1 to 100 nm, have been used to create unique devices at the nanoscale level possessing novel physical and chemical functional properties (Colvin, 2003, Oberdörster, 2004). Although nanomaterials are currently being widely used in modern technology, there is a serious lack of information concerning the human health and environmental implications of manufactured nanomaterials. The major toxicological concern is the fact that some of the manufactured nanomaterials are redox active (Colvin, 2003), and some particles transport across cell membranes and especially into mitochondria (Foley et al., 2002). One of the few relevant studies was with single-wall carbon nanotubes in mice (Lam et al., 2004). Lam et al. (2004) demonstrated that carbon nanotube products induced dose-dependent epithelioid granulomas in mice and, in some cases, interstitial inflammation in the animals of the 7-day post-exposure groups. The recent study by Oberdörster (2004) indicated that nanomaterials (Fullerences C60) induced oxidative stress in a fish model. Although limited studies have been conducted on the toxicity of nanoparticles, there are no reports on the use of in vitro models to evaluate potential toxicity screening of nanomaterials. The BRL 3A immortal rat liver cell line was selected in the present study as a convenient in vitro model to assess nanocellular toxicity. This cell line has been well characterized for its relevance to toxicity models (Boess et al., 2003). In vivo exposure to nanoparticles is likely to have potential impact on the liver since exposure to these particles is likely to occur through ingestion and clearance by the liver (Jani et al., 1990). The toxicity end points (MTT, LDH, ROS and GSH) that were selected in the current study represent vital biological functions of the mammalian system as well as provide a general sense of toxicity in a relatively short time. The results described in this paper provide a range of doses that were toxic to these cultured cells and data pointing to a general mechanism of nanoparticle toxicity. Since little information is available on nanomaterial toxicity, simple in vitro toxicity models and general toxicity end points are likely to assist in mechanistic events after exposure and subsequent toxicity risk assessment of nanomaterials.

Section snippets

Chemicals

The test materials silver (Ag; 15, 100 nm), molybdenum (MoO3; 30, 150 nm), aluminum (Al; 30, 103 nm), iron oxide (Fe3O4; 30, 47 nm), manganese oxide (MnO2; 1–2 μm), and tungsten (W; 27 μm) were received from Air Force Research Laboratory, Brooks AFB, TX. Cadmium oxide (CdO-1000 nm) and titanium oxide (TiO2-40 nm) were purchased from Fluka Chemicals and Altair, Nanomaterials Inc., respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), β-nicotinamide-adenine

Results

The results demonstrated that exposure to Ag nanoparticles for 24 h resulted in concentration-dependent increase in LDH leakage and exhibited a significant (p < 0.05) cytotoxicity at 10–50 μg/ml (Fig. 1B). It was noted that there is a statistically significant difference between different silver particle sizes of 100 and 15 nm, where the 100 nm particles showed higher toxicity at 25 and 50 μg/ml. The results for LDH leakage for MoO3, Al, Fe3O4, MnO2, W, nanoparticles exposure did not produce

Discussion

The purpose of this investigation was to evaluate potential toxicity and the general mechanism involved in nanoparticle toxicity. To date there are very few studies directly or indirectly investigating the toxic effects of nanomaterials and no clear guidelines are presently available to quantify these effects. Recently, Lam et al. (2004) reported that nanotubes induced lung tissue damage in mice resulting in granulomas. Another report by Warheit et al. (2004) investigated acute lung toxicity

Acknowledgements

This work was supported by the Air Force Office of Scientific Research (AFOSR) Project (JON# 2312A211) and performed in conjunction with U.S. Air Force Contract F41624-96-C-9010 (ManTech/Geo-Centers Joint Venture). We are thankful to our Division Chief Col Riddle for his strong support and encouragement. The authors are thankful to Dr. Marie Claude-Hofmann, University of Dayton for helping fluorescence microscopic work.

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