Environ. Sci. Technol. 2009, 43, 948–953
Is Lipid Peroxidation Induced by the Aqueous Suspension of Fullerene C60 Nanoparticles in the Brains of Cyprinus carpio? N A O H I D E S H I N O H A R A , * ,† TAKERU MATSUMOTO,‡ MASASHI GAMO,† ARISA MIYAUCHI,† SHIGEHISA ENDO,† YOSHITAKA YONEZAWA,† AND JUNKO NAKANISHI† National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba-shi, Ibaraki 305-8569, Japan, and Mitsubishi Chemical Safety Institute Ltd., 1000 Kamoshida-cho, Aoba-ku, Yokohama-shi, Kanagawa 227-0033, Japan
Received August 4, 2008. Revised manuscript received November 6, 2008. Accepted November 10, 2008.
This study aimed to clarify whether fullerene C60 nanoparticles induced lipid peroxidation in Cyprinus carpio brains. A stable well-characterized aqueous suspension of C60 nanoparticle (diameter: 50th and 95th percentiles, 36 and 95 nm respectively) with 0.1% Tween80 solution was prepared by bead milling. Lipid hydroperoxides (LPO) were measured in vitro in homogenized fish brain tissues containing 33 µg/mg-protein dispersed C60 nanoparticles under light and dark conditions to verify the lipid peroxidation ability of C60 and the interference of light exposure by using a commercial assay kit. The LPO concentration significantly increased under the light condition but not under the dark condition. This suggests that C60 has the lipid peroxidation ability under light condition, and the light exposure that occurs during the dissection and preparation of fish brain samples containing C60 for the LPO assay interferes with the measurements of the in vivo LPO levels. Therefore, dissection and assay in the in vivo experiment were conducted under a yellow lamp or dark condition to avoid the interference of light. Moreover, the result of the in vitro test suggests that the LPO assay with irradiation might be a good method for detecting C60 in brain tissues. In the in vivo experiment, C. carpio was exposed to 4.5 mg/L nano C60 suspension for 48 h, following which the brain LPO concentration was measured. In the in vivo experiment, no fish died or exhibited abnormal symptoms during exposure. LPO assay of the C. carpio brain samples confirmed the absence of lipid peroxidation after exposure to 4.5 mg/L aqueous C60 nanoparticle suspension for 48 h. Additional LPO assay under irradiation showed that C60 did not reach the brain.
Introduction Fullerene C60, which possesses unique physical and chemical properties, is a candidate substance in many nanotechnology * Corresponding author phone: +81-29-861-8030; fax: +81-29861-8411; e-mail:
[email protected]. † National Institute of Advanced Industrial Science and Technology. ‡ Mitsubishi Chemical Safety Institute Ltd. 948
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 3, 2009
applications in the industrial and medical fields, such as energy conversion and drug delivery. The fact that nanomaterials have the potential to harm is cause for concern because the small size of these particles facilitates their easy transfer into and reaction with the human body. In medaka (1) and rats (2), nanoparticles, albeit very few, penetrate the blood-brain barrier and reach the brain. Smaller aggregates of C60 nanoparticles (∼2 nm) prepared by stirring in water had greater antibacterial activity against Bacillus subtilis than larger aggregates of C60 (142 nm) (3). In addition, it has been reported that C60 nanoparticles possibly induce lipid peroxidation. Oberdo¨ster reported that exposure for 48 h to a 0.5 mg/L aqueous suspension of C60 nanoparticles (nano C60 suspension) (30∼100 nm), which was prepared using tetrahydrofuran (THF) (THF-nC60), induced significant lipid peroxidation in the brains of juvenile largemouth bass (Micropterus salmoides) (4). Sayes et al. reported that THF-nC60 is cytotoxic because of its ability to induce lipid peroxidation (5). Several methods for the preparation of nano C60 suspension were considered for the evaluation of the toxicity of C60 particles toward aquatic organisms and rodents. In some cases, a solution of C60 in an organic solvent, such as ethanol (6) and THF (7) is mixed with water, and the organic solvent is then removed from the mixture. In others, water containing C60 particles is stirred for 2 weeks or longer (8, 9), or C60 particles are ground with Tween80 (10). Lipid peroxidation was induced by nano C60 suspension in previous studies; however, this might not be attributable to C60 itself because THF-nC60 was reported to be inappropriate for some toxicity tests of C60. For example, THF-nC60 is more toxic toward bacteria (11), daphnia (12, 13), and larval zebrafish (14) than a nano C60 suspension prepared by stirring and sonication. The toxic effects of THF-nC60 on aquatic organisms were considered to be mediated by a THF-degradation product (γ-butyrolactone) rather than by fullerene C60 (14). In both in vitro and in vivo tests, the irradiation of samples containing C60 during dissection, assay preparation, and analysis may induce some unexpected effects attributable to the property of C60 as follows. It is known that C60 is a photosensitizer, and photoexcited C60 generates superoxide anions (O2-) upon irradiation (15). Many in vitro tests showed that fullerene and its derivatives acquire cytotoxicity on irradiation, but the underlying mechanism remains unresolved (16-18). The characterization of the size and/or stability of C60 nanoparticles in suspension is very important to evaluate their toxicity; however, studies on the toxicity of C60 have not described in sufficient detail the characteristics of the test samples used. For example, the particle size was not stated or only the particle size reported by the manufacturer was mentioned. Particle size can change during the preparation of the suspension, dilution, and exposure. As mentioned above, some adverse effects mentioned in these reports could be induced by factors other than C60, such as impurities introduced due to the method used to prepare the nano C60 suspension, altered activity due to the light conditions during the test, and stability/reliability of particle size. Thus, the purpose of our study is to determine whether C60 nanoparticles themselves induce lipid peroxidation in fish brains without the addition of any organic solvents such as THF. To counter the effects of irradiation and stability/reliability of C60 particle size, a stable and wellcharacterized nano C60 suspension was prepared and treated under controlled light conditions in this study. 10.1021/es802182f CCC: $40.75
2009 American Chemical Society
Published on Web 12/23/2008
TABLE 1. Conditions of Water and Nano C60 Suspension in the Exposure Test particle size [nm] test group
observed time [h]
temperature [°C]
DO [mg/L]
pH
control
0 24 48
23.9 24.1 24.0
8.3 6.8 6.8
7.7 7.3 7.3
exposure
0 24 48
23.7 24.1 24.0
8.2 6.3 6.0
7.7 7.4 7.3
In this manuscript, the following experiments are described. (i) Stable nano C60suspension (diameter of 95% particles 99.5%; Frontier Carbon Co. Ltd., Japan) was mixed with a few milliliters of 0.1% Tween80 (MP Biomedicals LLC, U.S.) aqueous solution and pounded in an agate mortar for 30 min under nitrogen purge to avoid C60O formation. (ii) The pounded solution was diluted with approximately 500 mL 0.1% Tween80 aqueous solution. (iii) C60 in the solution was pulverized and dispersed in Ultra Apex Mill (UAM-015; Kotobuki Industries Co. Ltd., Japan) with 50 µm zirconium beads (400 g) under nitrogen purge at approximately 4 L/min for 30 min (agitation rate: 79.1 m/s). (iv) The nano C60 suspension was separated by centrifugation (MX300; Tomy Seiko Co. Ltd., Japan) at 8000g for 60 min. (v) Finally, the supernatant fluid was pipetted and used to prepare the test stock suspension. The water used during this preparation was deionized and deaerated before use in the above process. The test solution for the exposure test was prepared by diluting the stock suspension. For the detection test of C60 in brain tissue, we prepared a highly concentrated suspension by repeated ultrafiltration with centrifugal filter devices (Centriplus YM-100; Millipore Co., U.S.) at 3000g for 30 min. Characterization of Nano C60 Suspension. Size distributions of the C60 nanoparticles in suspension were determined by dynamic light scattering (DLS) (Zetasizer nano-ZS; Malvern Instruments Ltd., UK). The ζ potentials of the aqueous suspension were measured with the same instrument. The structure of C60 nanoparticles in the aqueous suspension was observed through a transmission electron microscope (TEM) (TEM-1010; JEOL Datum Ltd., Japan). The concentration of the aqueous suspension was determined as follows.
concentration [mg/L]
median
95th percentile
ζ-potential [mV]
4.60 4.43 4.32
35.0 ( 1.8 38.4 ( 0.98 38.2 ( 1.8
90.9 ( 1.9 93.4 ( 0.75 102 ( 1.5
-17.7 ( 1.2 -20.2 ( 0.36 -17.8 ( 2.1
First, 10 mL aqueous suspension was evaporated to dryness with a rotatory evaporator, and the dried residue was dissolved in 10 mL toluene (HPLC grade, Wako Pure Chemicals Co. Ltd., Japan) with ultrasonication. The concentration of C60 in the toluene solution was analyzed using high-performance liquid chromatography (HPLC) (HP1100; Hewlett-Packard Co., U.S.) with a photodiode array detector (wavelength, 334 nm). A packed Kaseisorb LC ODS-60-5 column (Tokyo Kasei Kogyo Co. Ltd.; length: 150 mm × inner diameter: 4.6 mm, particle size: 5 µm, and pore size: 6 nm) was used for the separation. A mixture of 40% toluene and 60% methanol was used as a mobile phase at a flow rate of 1 mL/min. The injection volume for each analysis was 10 µL. The column temperature was set at 40 °C. Fish. Although a standard method for the identification of lipid peroxidation in fish has not been established, the common carp is one of the fishes recommended in the Organization for Economic Cooperation and Development (OECD) Guidelines for the Testing of Chemicals, no. 203 “Fish, Acute Toxicity Test” (20) for determining the median lethal concentration. In addition, the common carp is one of the most common fishes in the Japanese environment and is often used for toxicity tests in Japan (21). Therefore, we used the common carp Cyprinus carpio to determine the effects of C60 on fish brains. Juvenile common carp C. carpio was purchased from Niikura Fish Farm in Isehara, Kanagawa, Japan in November 2006 for the in vitro experiment and in June 2007 for the exposure test, and maintained in the Yokohama laboratory at Mitsubishi Chemical Safety Institute Ltd. Fish were reared in an 120 L glass aquarium with flow-through of dechlorinated water at 24 ( 1 °C under a 16:8 h light:dark cycle. They were fed 2% of their body weight per day of BabyGold (Kyorin Co. Ltd., Japan) until 24 h before the beginning of the exposure test. The quality of dechlorinated water for the in vitro and exposure experiments conducted in 2006 and 2007 are as follows are shown in SI Table S1. In Vitro Experiment. Ten fish (average weight: 9.96 g, average length: 83.4 mm) were anaesthetized on ice, dissected, and weighed. Thereafter, the brains (average weight: 159 mg) were immediately frozen in liquid nitrogen and stored at -80 °C until the experiment. The brains were homogenized with 4 times their volume of 50 mM 2-(Nmorpholino) ethane sulfonic acid (MES; Dojindo Laboratories, Japan) buffer (pH 6.7) by using an electric homogenizer (Ultra-Turrax T-8; IKA-Werke GmbH & Co. KG, Germany). In total, four types of mixtures were tested (MES + Tween80, brain tissue + MES + Tween80, MES + C60 + Tween80, and brain tissue + MES + C60 + Tween80). Homogenized brain tissue (250 µL) or 50 mM MES (250 µL) was mixed with 250 µL nano C60 suspension (0.52 mg/mL; 0.1% Tween80) or 250 µL 0.1% Tween80 aqueous solution (N ) 6 for each sample). Next, three samples of each mixture were maintained under light (approximately 600 lx), while three other samples of each mixture were maintained under dark condition (
