Carbon fullerenes (C60s) can induce inflammatory responses in the lung of mice

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Abstract

Fullerenes (C60s) occur in the environment due to natural and anthropogenic sources such as volcanic eruptions, forest fires, and the combustion of carbon-based materials. Recently, production and application of engineered C60s have also rapidly increased in diverse industrial fields and biomedicine due to C60′ unique physico-chemical properties, so toxicity assessment on environmental and human health is being evaluated as a valuable work. However, data related to the toxicity of C60s have not been abundant up to now. In this study, we studied the immunotoxic mechanism and change of gene expression caused by the instillation of C60s. As a result, C60s induced an increase in sub G1 and G1 arrest in BAL cells, an increase in pro-inflammatory cytokines such as IL-1, TNF-α, and IL-6, and an increase of Th1 cytokines such as IL-12 and IFN-r in BAL fluid. In addition, IgE reached the maximum at 1 day after treatment in both BAL fluid and the blood, and decreased in a time-dependent manner. Gene expression of the MHC class II (H2-Eb1) molecule was stronger than that of the MHC class I (H2-T23), and an increase in T cell distribution was also observed during the experiment period. Furthermore, cell infiltration and expression of tissue damage related genes in lung tissue were constantly observed during the experiment period. Based on this, C60s may induce inflammatory responses in the lung of mice.

Introduction

Fullerene was discovered in 1985 by Robert Curl and is a carbon allotrope that is composed entirely of carbon. The most abundant form is buckminsterfullerene (C60) with 60 carbon atoms arranged in a spherical structure (Kroto et al., 1985). C60s occur in the environment due to natural and anthropogenic sources such as volcanic eruptions, forest fires, and the combustion of carbon-based materials (e.g., coal, soot) as MWCNT (multiwall carbon nanotube). Recently, production and application of engineered C60s have also rapidly increased in diverse industrial fields and biomedicine due to C60′ unique physico-chemical properties, so toxicity assessment on environmental and human health is being evaluated as more valuable work. In this respect, it is fortunate that full-scale exposure to the public has not been started yet, so the current study results can be directly fed back to the product development plan. Manufacturers can produce safer products without risks of waiting for toxicology studies to be finished after the products are widely marketed (Tsuda et al., 2008).

Currently, there are contradictory reports whether C60s act as a radical scavenger or as a generator. Krusic et al. characterized fullerenes as “radical sponges” in 1991, and many researchers supported the hypothesis by publishing other results. However, others have reported that C60s induced damage in tissues and cells through lipid peroxidation and oxidative stress in vivo and in vitro (Krusic et al., 1991, Misirkic et al., 2009, Sayes et al., 2005, Oberdorster, 2004, Hu et al., 2007, Zhu et al., 2006, Zhu et al., 2008, Usenko et al., 2008).

In addition, particles stimulate the immune system in the body. C60s induced MHC (major histocompatibility complex)-mediated immunotoxicity under the systemic inhalation exposure condition and a significant increase of protein in BAL (Bronchoalveolar Lavage) fluid due to a nose-only exposure system (Baker et al., 2008, Fujita et al., 2009). In this study, we investigated the imunotoxic mechanism of C60s in mice intratracheally instilled with this material. Furthermore, to give useful information concerning the hazards of nanoparticles to the public, characterization related to physico-chemical properties of nanoparticles such as morphology, size, and surface chemistry had to be performed before the toxicity test. C60s have a low solubility of about 1.3 × 10 11 μg/L in water, and form water-stable aggregates with diameters up to 500 nm (Spohn et al., 2009). Toxicologists get water-soluble suspension of C60s by utilizing various organic solvents, including THF, benzene, toluene, and DMSO to overcome this problem. Prior studies suggest that this solvent can alter the physico-chemical properties and the toxicity of C60s. Therefore, we measured the size, conductance, mobility, and surface charge of C60s, which was modified by toluene as the solvent for PBS-soluble suspension, and confirmed the shape, aggregation, and agglomeration state using TEM prior to the toxicity test; we also utilized the solvent as control vehicle.

Section snippets

Animals

ICR male mice (25 ± 1 g) were purchased from Orient-Bio Animal Company (Seongnam, Gyunggi-do, Korea) and were allowed to adapt to the animal room conditions prior to initiation of the study. The environmental conditions were a temperature of 23 ± 1 °C, relative humidity of 55 ± 5%, and a 12 h light/dark cycle. All the animals used in this study were cared for in accordance with the principles outlined in the “Guide for the Care and Use of Laboratory Animals” issued by the Animal Care and Use

Stability of C60s in PBS

Before instillation, we investigated the stability of C60s in PBS. Particle size was 46.7 ± 18.6 nm (Fig. 1A), and surface charge was − 30.24 mV, conductance was 22 μS, and average mobility was − 2.36 (μ/s)/(V/cm) (Fig. 1B). TEM micrographs confirmed that the C60s were spherical in shape and were mildly agglomerated in PBS (Fig. 1C).

Cell cycle of BAL cell

Distribution of the G1 stage increased to 95.04%, 96.89%, 97.2%, and 97.29% at days 1, 7, 14, and 28 after treatment with 2 mg/kg C60s, respectively, whereas that of

Discussion

The carbon atoms in C60 together join to form a hexagonal structure resembling a soccer ball. Now, it is known that C60s have electron-donating or -accepting unique properties, but it is not clear yet whether C60s act as an anti- or pro-oxidant in biological systems (Zhu et al., 2006, Ryan et al., 2007). And, the organic solvent utilized to prepare water-soluble suspension can modify the properties of C60s and the modified C60s can alter the toxicity of pristine C60s. Sayes et al. investigated

Acknowledgment

This work was supported by the Ministry of Environment as the Eco-technopia 21 project.

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