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Toxicity of an engineered nanoparticle (fullerene, C60) in two aquatic species, Daphnia and fathead minnow
MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 62 (2006) S5–S9 www.elsevier.com/locate/marenvrev
Short communication
Toxicity of an engineered nanoparticle (fullerene, C60) in two aquatic species, Daphnia and fathead minnow q Shiqian Zhu a, Eva Oberdo¨rster a
b,c
, Mary L. Haasch
a,*
Environmental Toxicology Research Program, National Center for Natural Products Research, Department of Pharmacology, School of Pharmacy, The University of Mississippi, 347 Faser, University, MS 38677-1848, USA b Department of Biology, Southern Methodist University, TX, USA c Duke University Marine Laboratory, Beaufort, NC, USA
Abstract Water-soluble fullerene (nC60) has been shown to induce lipid peroxidation (LPO) in brain of juvenile largemouth bass (LMB, Micropterus salmoides) [Oberdo¨rster, E., 2004. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in brain of juvenile largemouth bass. Environ. Health Persp. 112, 1058–1062]; and upregulate genes related to the inflammatory response and metabolism, most notably CYP2K4 [Oberdo¨rster, G., Oberdo¨rster, E., Oberdo¨rster, J., 2005. Nanotoxicology: an emerging discipline evolving from 116 studies of ultrafine particles. Environ. Health Persp. 113, 823–839]. The initial study in LMB was performed using tetrahydrofuran (THF)-solubilized nC60, although C60 can also be solubilized by stirring in water. The current study investigates differences in acute toxicity to Daphnia magna between THF-solubilized and water-stirred-nC60 as a range-find for further assays in adult male fathead minnow (FHM, Pimephales promelas). The daphnia 48-h LC50 for THF-nC60 was at least one order of magnitude less (0.8 ppm) than that for waterstirred-nC60 (>35 ppm). FHM were dosed with either 0.5 ppm of THF- or water-stirred-nC60 for 48 h. There was 100% mortality in the THF-nC60-exposed fish between 6 and 18 h, while the q Supported by the Environmental Toxicology Research Program, National Center for Natural Products Research, The University of Mississippi, MS, USA to S.Z. and M.L.H.; and the Lonestar Nanotechnology Fund to E.O. * Corresponding author. Address: USEPA Mid-Continent Ecology Division, Molecular and Cellular Mechanisms Research Branch, 6201 Congdon Boulevard, Duluth, MN 55804, USA; Tel.: +1 218 529 5172; fax: +1 218 529 5003. E-mail addresses: [email protected], [email protected] (M.L. Haasch).
0141-1136/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2006.04.059
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S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
water-stirred-nC60-exposed fish showed no obvious physical effects after 48 h. Water-stirred-nC60 elevated LPO in brain, significantly increased LPO in gill, and significantly increased expression of CYP2 family isozymes in liver as compared to control fish. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Nanotoxicology; Fullerene; CYP2; Lipid peroxidation; LC50
Engineered nanoparticles (NP) are being used in a variety of products from sporting equipment to cosmetics; clothing to solar cells. Carbon NP such as C60 are lipophilic, which allows them to interact with membranes of all types (Foley et al., 2002; Kamat et al., 2000). Due to the interesting electron-donating and -accepting properties of C60, it has been found that modified C60 can release oxyradicals in in vitro systems (Kamat et al., 2000; Yamakoshi et al., 2003), or act as an oxyradical scavenger (Lee et al., 2000). It is difficult to predict whether fullerene will act as an anti- or pro-oxidant in vivo. Un-modified C60 is not immediately water soluble, and there are two methods of solubilization. Both methods form stable aggregates of C60 with diameters ranging from 10 to 200 nm, termed nano-C60 or nC60. The clusters contain neat C60 in the centers, surrounded by partially hydroxylated C60 on the outside, which make up less than 1% of the total C60 in solution (Fortner et al., 2005). In the THF-method, the C60 is first solubilized in tetrahydrofuran (THF), then mixed with water, and finally THF is evaporated away. This is a rapid method, but a residual amount of THF may be trapped in the center of the aggregates (Fortner et al., 2005), which likely leads to the differences in toxicity seen in this study. The water-stirred method takes more time (stirring for weeks) but is environmentally more relevant as it mimics wave movement and does not contain organic solvents (for details on the methods see Oberdo¨rster, 2005). Fullerene (99.5% pure) was purchased from SES Research (Houston, TX) and was solubilized in either THF or water-only using standard protocols (Oberdo¨rster, 2005). As a range-find assay, acute toxicity of THF- and water-stirred-nC60 was determined in a 48 h LC50 assay on Daphnia magna using the standard US EPA protocol for static renewal testing (EPA, 1994). The 48 h LC50 for THF-nC60 was considerably lower (more toxic) than the 48 h LC50 for water-stirred-nC60 (0.8 ppm compared to >35 ppm, respectively; Fig. 1). Adult male fathead minnows (FHM) were dosed individually with either (a) reconstituted hard water (RHW), (b) 0.5 ppm water-stirred-nC60 in RHW, or (c) 0.5 ppm THFnC60 in RHW (n = 10 controls, n = 5 each of the two nC60 exposures) in 1 L aquaria for 48 h, with a 50% water change at 24 h. Fish were euthanized using MS-222, and tissues from each fish were flash frozen. Lipid peroxidation (LPO) was determined as previously described using the malonaldehyde method in brain and gill (Oberdo¨rster, 2004). Statistical analysis was done using SYSTAT 8.0, using student t-test. Immunodetection was performed using rabbit anti-trout CYP2K1 and CYP2M1 polyclonal antibodies (generous gift from Donald R. Buhler, Oregon State University) essentially as previously described (Haasch, 2002). Following densitometry, one outlier control value was removed and the data were log-transformed prior to statistical analyses using GraphPad Prism 4.0 (GraphPad Software, Inc, San Diego, CA). Unlike the previous studies in largemouth bass (LMB) using THF-nC60 (Oberdo¨rster, 2004), FHM exposed to 0.5 ppm of THF-nC60 died within 18 h of exposure. Water-stirred-
S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
S7
Cumulative mortality of D. magna exposed to nC60 (THF method)
A
% mortality
100 Control 5 ppb 10 ppb 25 ppb 100 ppb 500 ppb 1000 ppb 2000 ppb
80 60 40 20 0 0
1
2
3
4
5
6
7
8
day of exposure B
Cumulative mortality in D. magna exposed to nC60 (water-stirred method) 100
Control 0.5 ppm 1ppm 2.5 ppm 5 ppm 35 ppm
% mortality
80 60 40 20 0
1
2
4 5 3 day of exposure
6
7
8
Fig. 1. Mortality curves for Daphnia magna exposed to either THF- (A) or water-stirred-nC60 (B). The THF-nC60 is at least one order of magnitude more toxic than the water-stirred-nC60, highlighting the importance of relevant preparation techniques for aquatic toxicity testing. The 48 h LC50 for THF-nC60 was calculated to be 0.8 ppm (800 ppb), and the 48 h LC50 for water-stirred-nC60 was >35 ppm.
nC60 exposed FHM brain tissue exhibited elevated, although not statistically significant, LPO, while in gill there was statistically higher LPO (data not shown). Hepatic levels of some CYP2-like isozymes were significantly increased (Fig. 2). One induced protein was immunoreactive with anti-CYP2K1 (53 kDa), and two proteins were immunoreactive with anti-CYP2M1, of which the higher MW protein (53 kDa), was significantly induced. These probable CYP2-family isozymes have not previously been described in FHM. This is the first in vivo example, in any species, of changes in P450 protein expression due to nC60 exposure. The method of nC60 preparation can affect the toxicity of fullerene, with THF-nC60 more toxic than water-stirred-nC60 in both daphnia and FHM. Anecdotal evidence and data not presented here shows that even sonicating C60 to get it into solution more quickly increases the toxicity of nC60. It is therefore critical to use environmentally-relevant doses and preparation techniques. Exposure to water-stirred-nC60 can induce CYP2 isozymes in exposed fish at relatively low levels (0.5 ppm) and within a short time (48 h). Fullerene can increase LPO in fish exposed via the water column, similar to what has been found previously (Oberdo¨rster,
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S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
Fig. 2. Significant induction of CYP2-family isozymes in male fathead minnow by 0.5 ppm, 48 h, water-stirrednC60 exposure. (A) CYP2K1-like protein (53 kDa), (B) CYP2M1-like Protein 1 (53 kDa), (C) CYP2M1-like Protein 2 (48 kDa).
S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
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2004). Up-regulated CYP2 isozymes may function in metabolizing fullerene or in repairing LPO. Future research needs to address uptake, distribution, biotransformation and the pathway-specific changes in gene expression due to exposure to nanomaterials. Acknowledgments E.O. solubilized the fullerene, performed the acute toxicity assays in Daphnia magna, exposed the FHM in her laboratory, and performed the LPO assays. S.Z. performed the FHM immunodetection and analysis in the laboratory of M.L.H. References EPA, 1994. 10-day chronic – Daphnia magna or Daphnia pulex; SOP # 2028. Available from:. Foley, S., Crowley, C., Smaihi, M., Bonfils, C., Erlanger, B., Seta, P., Larroque, C., 2002. Cellular localisation of a water-soluble fullerene derivative. Biochem. Biophys. Res. Commun. 294, 116–119. Fortner, J.D., Lyon, Dy, Sayes, C.M., Boyd, A.M., Falkner, J., Hotze, E., Alemany, L., Tao, Y., Ausman, K., Colvin, V., Hughes, J., 2005. C60 in water: nanocrystal formation and microbial response. Environ. Sci. Technol. 39, 4307–4316. Haasch, M.L., 2002. Effects of vehicle, diet and gender on the expression of PMP70- and CYP2K1/2M1-like proteins in the mummichog. Mar. Environ. Res. 54, 297–301. Kamat, J., Devasagayam, T., Priyadarsini, K., Mohan, H., 2000. Reactive oxygen species mediated membrane damage induced by fullerene derivatives and its possible biological implications. Toxicology 155, 55–61. Lee, Y., Chiang, L., Chen, W., Hsu, H., 2000. Water-soluble hexasulfobutyl[60]fullerene inhibit low-density lipoprotein oxidation in aqueous and lipophilic phases. Proc. Soc. Exp. Biol. Med. 224, 69–75. Oberdo¨rster, E., 2004. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in brain of juvenile largemouth bass. Environ. Health Persp. 112, 1058–1062. Oberdo¨rster, E., 2005. Informal information on the toxicity of engineered nanomaterials. Available from: . Oberdo¨rster, G., Oberdo¨rster, E., Oberdo¨rster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Persp. 113, 823–839. Yamakoshi, Y., Umezawa, N., Ryu, A., Arakane, K., Miyata, N., Goda, Y., Masumizu, T., Nagano, T., 2003. Active oxygen species generated from photoexcited fullerene (C60) as potential medicines: O2 * versus 1O2. J. Am. Chem. Soc. 125, 12803–12809.
Short communication
Toxicity of an engineered nanoparticle (fullerene, C60) in two aquatic species, Daphnia and fathead minnow q Shiqian Zhu a, Eva Oberdo¨rster a
b,c
, Mary L. Haasch
a,*
Environmental Toxicology Research Program, National Center for Natural Products Research, Department of Pharmacology, School of Pharmacy, The University of Mississippi, 347 Faser, University, MS 38677-1848, USA b Department of Biology, Southern Methodist University, TX, USA c Duke University Marine Laboratory, Beaufort, NC, USA
Abstract Water-soluble fullerene (nC60) has been shown to induce lipid peroxidation (LPO) in brain of juvenile largemouth bass (LMB, Micropterus salmoides) [Oberdo¨rster, E., 2004. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in brain of juvenile largemouth bass. Environ. Health Persp. 112, 1058–1062]; and upregulate genes related to the inflammatory response and metabolism, most notably CYP2K4 [Oberdo¨rster, G., Oberdo¨rster, E., Oberdo¨rster, J., 2005. Nanotoxicology: an emerging discipline evolving from 116 studies of ultrafine particles. Environ. Health Persp. 113, 823–839]. The initial study in LMB was performed using tetrahydrofuran (THF)-solubilized nC60, although C60 can also be solubilized by stirring in water. The current study investigates differences in acute toxicity to Daphnia magna between THF-solubilized and water-stirred-nC60 as a range-find for further assays in adult male fathead minnow (FHM, Pimephales promelas). The daphnia 48-h LC50 for THF-nC60 was at least one order of magnitude less (0.8 ppm) than that for waterstirred-nC60 (>35 ppm). FHM were dosed with either 0.5 ppm of THF- or water-stirred-nC60 for 48 h. There was 100% mortality in the THF-nC60-exposed fish between 6 and 18 h, while the q Supported by the Environmental Toxicology Research Program, National Center for Natural Products Research, The University of Mississippi, MS, USA to S.Z. and M.L.H.; and the Lonestar Nanotechnology Fund to E.O. * Corresponding author. Address: USEPA Mid-Continent Ecology Division, Molecular and Cellular Mechanisms Research Branch, 6201 Congdon Boulevard, Duluth, MN 55804, USA; Tel.: +1 218 529 5172; fax: +1 218 529 5003. E-mail addresses: [email protected], [email protected] (M.L. Haasch).
0141-1136/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2006.04.059
S6
S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
water-stirred-nC60-exposed fish showed no obvious physical effects after 48 h. Water-stirred-nC60 elevated LPO in brain, significantly increased LPO in gill, and significantly increased expression of CYP2 family isozymes in liver as compared to control fish. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Nanotoxicology; Fullerene; CYP2; Lipid peroxidation; LC50
Engineered nanoparticles (NP) are being used in a variety of products from sporting equipment to cosmetics; clothing to solar cells. Carbon NP such as C60 are lipophilic, which allows them to interact with membranes of all types (Foley et al., 2002; Kamat et al., 2000). Due to the interesting electron-donating and -accepting properties of C60, it has been found that modified C60 can release oxyradicals in in vitro systems (Kamat et al., 2000; Yamakoshi et al., 2003), or act as an oxyradical scavenger (Lee et al., 2000). It is difficult to predict whether fullerene will act as an anti- or pro-oxidant in vivo. Un-modified C60 is not immediately water soluble, and there are two methods of solubilization. Both methods form stable aggregates of C60 with diameters ranging from 10 to 200 nm, termed nano-C60 or nC60. The clusters contain neat C60 in the centers, surrounded by partially hydroxylated C60 on the outside, which make up less than 1% of the total C60 in solution (Fortner et al., 2005). In the THF-method, the C60 is first solubilized in tetrahydrofuran (THF), then mixed with water, and finally THF is evaporated away. This is a rapid method, but a residual amount of THF may be trapped in the center of the aggregates (Fortner et al., 2005), which likely leads to the differences in toxicity seen in this study. The water-stirred method takes more time (stirring for weeks) but is environmentally more relevant as it mimics wave movement and does not contain organic solvents (for details on the methods see Oberdo¨rster, 2005). Fullerene (99.5% pure) was purchased from SES Research (Houston, TX) and was solubilized in either THF or water-only using standard protocols (Oberdo¨rster, 2005). As a range-find assay, acute toxicity of THF- and water-stirred-nC60 was determined in a 48 h LC50 assay on Daphnia magna using the standard US EPA protocol for static renewal testing (EPA, 1994). The 48 h LC50 for THF-nC60 was considerably lower (more toxic) than the 48 h LC50 for water-stirred-nC60 (0.8 ppm compared to >35 ppm, respectively; Fig. 1). Adult male fathead minnows (FHM) were dosed individually with either (a) reconstituted hard water (RHW), (b) 0.5 ppm water-stirred-nC60 in RHW, or (c) 0.5 ppm THFnC60 in RHW (n = 10 controls, n = 5 each of the two nC60 exposures) in 1 L aquaria for 48 h, with a 50% water change at 24 h. Fish were euthanized using MS-222, and tissues from each fish were flash frozen. Lipid peroxidation (LPO) was determined as previously described using the malonaldehyde method in brain and gill (Oberdo¨rster, 2004). Statistical analysis was done using SYSTAT 8.0, using student t-test. Immunodetection was performed using rabbit anti-trout CYP2K1 and CYP2M1 polyclonal antibodies (generous gift from Donald R. Buhler, Oregon State University) essentially as previously described (Haasch, 2002). Following densitometry, one outlier control value was removed and the data were log-transformed prior to statistical analyses using GraphPad Prism 4.0 (GraphPad Software, Inc, San Diego, CA). Unlike the previous studies in largemouth bass (LMB) using THF-nC60 (Oberdo¨rster, 2004), FHM exposed to 0.5 ppm of THF-nC60 died within 18 h of exposure. Water-stirred-
S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
S7
Cumulative mortality of D. magna exposed to nC60 (THF method)
A
% mortality
100 Control 5 ppb 10 ppb 25 ppb 100 ppb 500 ppb 1000 ppb 2000 ppb
80 60 40 20 0 0
1
2
3
4
5
6
7
8
day of exposure B
Cumulative mortality in D. magna exposed to nC60 (water-stirred method) 100
Control 0.5 ppm 1ppm 2.5 ppm 5 ppm 35 ppm
% mortality
80 60 40 20 0
1
2
4 5 3 day of exposure
6
7
8
Fig. 1. Mortality curves for Daphnia magna exposed to either THF- (A) or water-stirred-nC60 (B). The THF-nC60 is at least one order of magnitude more toxic than the water-stirred-nC60, highlighting the importance of relevant preparation techniques for aquatic toxicity testing. The 48 h LC50 for THF-nC60 was calculated to be 0.8 ppm (800 ppb), and the 48 h LC50 for water-stirred-nC60 was >35 ppm.
nC60 exposed FHM brain tissue exhibited elevated, although not statistically significant, LPO, while in gill there was statistically higher LPO (data not shown). Hepatic levels of some CYP2-like isozymes were significantly increased (Fig. 2). One induced protein was immunoreactive with anti-CYP2K1 (53 kDa), and two proteins were immunoreactive with anti-CYP2M1, of which the higher MW protein (53 kDa), was significantly induced. These probable CYP2-family isozymes have not previously been described in FHM. This is the first in vivo example, in any species, of changes in P450 protein expression due to nC60 exposure. The method of nC60 preparation can affect the toxicity of fullerene, with THF-nC60 more toxic than water-stirred-nC60 in both daphnia and FHM. Anecdotal evidence and data not presented here shows that even sonicating C60 to get it into solution more quickly increases the toxicity of nC60. It is therefore critical to use environmentally-relevant doses and preparation techniques. Exposure to water-stirred-nC60 can induce CYP2 isozymes in exposed fish at relatively low levels (0.5 ppm) and within a short time (48 h). Fullerene can increase LPO in fish exposed via the water column, similar to what has been found previously (Oberdo¨rster,
S8
S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
Fig. 2. Significant induction of CYP2-family isozymes in male fathead minnow by 0.5 ppm, 48 h, water-stirrednC60 exposure. (A) CYP2K1-like protein (53 kDa), (B) CYP2M1-like Protein 1 (53 kDa), (C) CYP2M1-like Protein 2 (48 kDa).
S. Zhu et al. / Marine Environmental Research 62 (2006) S5–S9
S9
2004). Up-regulated CYP2 isozymes may function in metabolizing fullerene or in repairing LPO. Future research needs to address uptake, distribution, biotransformation and the pathway-specific changes in gene expression due to exposure to nanomaterials. Acknowledgments E.O. solubilized the fullerene, performed the acute toxicity assays in Daphnia magna, exposed the FHM in her laboratory, and performed the LPO assays. S.Z. performed the FHM immunodetection and analysis in the laboratory of M.L.H. References EPA, 1994. 10-day chronic – Daphnia magna or Daphnia pulex; SOP # 2028. Available from: