Ecotoxicological investigation of CeO2 and TiO2 nanoparticles on the soil nematode Caenorhabditis elegans using gene expression, growth, fertility, and survival as endpoints

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Abstract

In this study, the potential harmful effect of cerium dioxide (CeO2), and titanium dioxide (TiO2) nanoparticles on the environment was investigated using Caenorhabditis elegans ecotoxicity tests. Multiple toxic endpoints, such as stress-response gene expression, growth, fertility, and survival, were analyzed in C. elegans, in response to the CeO2 and TiO2 exposure. To investigate relationship between sizes of nanoparticles and toxicity, C. elegans were exposed to nanoparticles to the different sizes of nanoparticles (15, 45 nm for CeO2 and 7, 20 nm for TiO2). An increase in the expression of the cyp35a2 gene, decrease in fertility and survival parameters were observed in the 15 and 45 nm of CeO2 and in the 7 nm of TiO2 nanoparticles exposed to C. elegans. Gene knock-down experiment using RNA interference (RNAi) suggested that physiological level disturbances may be related with the cyp35a2 gene expression. Smaller sized nanoparticles (7 nm of TiO2 and 15 nm of CeO2) seemed to be more toxic than larger sized ones (20 nm of TiO2 and 45 nm of CeO2) on the observed toxicity. The size-dependent effect in CeO2 and TiO2 nanoparticles-induced toxicity needs to be investigated under more detailed experimental settings with the various sizes of nanoparticles. Further studies on the mechanism by which CeO2 and TiO2 nanoparticles affect cyp35a2 gene expression, fertility, and survival are warranted to better understand the CeO2 and TiO2 nanoparticles-induced ecotoxicity in C. elegans, as are studies with the causal relationships between these parameters. Overall results suggest that CeO2 and TiO2 nanoparticles have a potential for provoking ecotoxicity on C. elegans and the data obtained from this study can comprise a contribution to knowledge of the ecotoxicology of nanoparticles in C. elegans, about which little data are available.

Introduction

Nanoparticles toxicity studies are among the fastest growing areas of environmental toxicology research, because of the fast applications of the nanotechnology in a wide variety of fields (Zheng et al., 2005, Nohynek et al., 2007, Rogueda and Traini, 2007). The potential health and environmental risks related to the widespread production and use of the nanomaterials created by these technologies need to be investigated in terms of their toxic mechanism as well as their long term consequences (Hoet et al., 2004, Oberdorster et al., 2005, Nel et al., 2006, Handy et al., 2008). Nanoparticles exposure in the environment may cause a physiological response in certain organisms, which alter their fitness, and ultimately lead to population/community level changes. Most of the current literature on the toxicity of nanoparticles comes from mammalian studies on respiratory exposure, or from in vitro assays with mammalian cells (Xiao-Feng et al., 2005, Donaldson, 2006, Cha and Myung, 2007, Handy and Shaw, 2007, Warheit et al., 2007). Ecotoxicological studies with nanoparticles are currently increasing, with most focusing on aquatic organisms (Hund-Rinke and Simon, 2006, Lovern and Klaper, 2006, Federici et al., 2007, Lovern et al., 2007, Smith et al., 2007, Fujiwara et al., 2008, Blaise et al., 2008, Velzeboer et al., 2008). Few studies have been performed on terrestrial organisms (Jemec et al., 2008, Wang et al., 2009). In the current study, ecotoxicological assessments of nanoparticles were conducted on the soil nematodes, particularly Caenorhabditis elegans, using multiple toxic endpoints.

C. elegans, a free-living nematode that lives mainly in the liquid phase of soils, is the first multicellular organism to have its genome completely sequenced. The genome showed an unexpectedly high level of conservation with the vertebrate genome, which makes C. elegans an ideal system for biological studies, such as those in genetics, molecular biology, and development biology (Bettinger et al., 2004, Leacock and Reinke, 2006, Schafer, 2006, Schroeder, 2006). C. elegans is also a good animal model for the study of ecotoxicology. Due to its abundance in soil ecosystems, its convenient handling in the laboratory, and its sensitivity to different kinds of stresses, C. elegans is frequently used in ecotoxicological studies utilizing various exposure media, including soil and water (Williams and Dusenbery, 1990, Peredney and Williams, 2000, Boyd and Williams, 2003, Roh et al., 2006, Roh et al., 2007).

Cerium dioxide (CeO2) and titanium dioxide (TiO2) were studied, as they are widely used manufactured nanomaterials. CeO2 is one of the most important rare-earth oxides that have been widely investigated due to its unique properties and multiple applications, such as acting as a three-way catalyst in vehicle emission-control systems, electrolyte materials of solid oxide fuel cells, and ultraviolet blocking materials (Yu et al., 2001, Corma et al., 2004). A potent photocatalyst, TiO2 has a potential for wide application in self-cleaning fabrics, paint, building materials, as well as, in pharmaceutical and cosmetic products (Gelis et al., 2003, Aitken et al., 2006).

The ecotoxicological assessment of CeO2 and TiO2 nanoparticles in C. elegans was conducted by investigating stress-response gene expressions, growth, fertility, and survival as toxic endpoints. To investigate relationship between the sizes of nanoparticles and toxicity, nanoparticles were exposed to C. elegans at a constant concentration (1 mg/L) with different sizes (15, 45 nm for CeO2 and 7, 20 nm for TiO2). Moreover, to test the ecotoxicological relevance of CeO2 and TiO2 nanoparticles-induced gene expression, integration of gene expression with the organism/population level endpoints was attempted using C. elegans functional genomics tool, RNAi.

Section snippets

CeO2 and TiO2 nanoparticles

CeO2 nanoparticles were synthesized by the supercritical synthesis method, as described previously (Park et al., 2008). The schematic diagram for the synthesis apparatus is shown in Supplementary Fig. 1. TiO2 nanoparticles were purchased form Sigma–Aldrich Chemical (St. Louis, MO, USA). To measure the surface area of the nanoparticles, the Branauer, Emmett and Teller (BET) method was used, employing a volumetric adsorption apparatus, BELSORP-mini II (BEL Japan Inc., Osaka, Japan). To

Results and discussions

The special properties of nanoparticles upon dispersion in aqueous media, such as, tendency to form large aggregates and poor solubility, hamper ecotoxicological studies. It has been recognized that results obtained with unstable nanoparticles dispersion cannot be fully representative of a monodispersed, non-aggregated nanoparticles system (Borm et al., 2006, Van Hoecke et al., 2008). As it is difficult to prepare stable, monodispersed, aqueous nanoparticles suspensions, special dispersion

Conclusions

Overall results suggest that CeO2 and TiO2 nanoparticles provoke ecotoxicity on C. elegans fertility and survival, which may be related with cyp35a2 gene expression. Further studies on the mechanism by which CeO2 and TiO2 nanoparticles affect cyp35a2 gene expression, fertility, and survival are warranted to better understand the CeO2 and TiO2 nanoparticles-induced ecotoxicity in C. elegans, as are studies with the causal relationships between these parameters. The genome wide transcription

Conflict of interest statement

We have nothing to declare in conflict of interests.

Acknowledgments

This work was accomplished with the generous support of the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) and Korea Ministry of Environment as “The Eco-technopia 21 project”.

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