Nanotoxicology, June 2012; 6(4): 381–398
Nano-sized CuO, TiO2 and ZnO affect Xenopus laevis development RENATO BACCHETTA1, NADIA SANTO2, UMBERTO FASCIO2, ELISA MOSCHINI3, STEFANO FREDDI4, GIUSEPPE CHIRICO4, MARINA CAMATINI3, & PARIDE MANTECCA3 1
Department of Biology, 2Interdepartmental Centre of Advanced Microscopy (CIMA), University of Milano, Milan, Department of Environmental Science, and 4Department of Physics, Research Centre POLARIS, University of Milano-Bicocca, Milan, Italy 3
(Received 27 October 2010; accepted 24 March 2011)
Abstract The teratogenic potential of commercially available copper oxide (CuO), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) was evaluated using the standardized FETAX test. After characterization of NP suspensions by TEM, DLS and AAS, histopathological screening and advanced confocal and energy-filtered electron microscopy techniques were used to characterize the induced lesions and to track NPs in tissues. Except for nCuO, which was found to be weakly embryolethal only at the highest concentration tested, the NPs did not cause mortality at concentrations up to 500 mg/L. However, they induced significant malformation rates, and the gut was observed to be the main target organ. CuO NPs exhibited the highest teratogenic potential, although no specific terata were observed. ZnO NPs caused the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues. TiO2 NPs showed mild embryotoxicity, and it is possible that this substance could be associated with hidden biological effects. Ions from dissolved nCuO contributed greatly to the observed embryotoxic effects, but those from nZnO did not, suggesting that their mechanisms of action may be different.
Keywords: Nanoparticles, metal oxides, FETAX, embryotoxicity, environmental risk
Introduction Nanotechnology is a field of increasing economic and scientific interest that continues to exhibit rapid development. New nanomaterials (NMs) are being manufactured and promptly used in a variety of applications, including cosmetics and personal care products, electronics, drug delivery systems, manufacturing technologies and paints (Ward and Kach 2009). Nanotechnology has been projected to become a $1 trillion market by 2015 (Nel et al. 2006), with an estimated annual production of approximately 60,000 tons of nanoparticles (NPs) by 2020 (Lewinski et al. 2008). This represents a market associated with great economic interest, which implies that there will be effects on the environment and, consequently, on humans due to exposure to NPs, for which few toxicity data are available. The lack of toxicological data available for NMs makes it difficult to predict the real risk associated with exposure to these substances and compels the
scientific community to undertake research to guarantee the sustainable development of this recently developed technology. The growing use of NPs in a number of applications in different fields is becoming increasingly worthy of attention from institutional organizations related to their possible effects on public health and ecosystems. During their life cycle, NPs will enter the environment, and experimental evidence has already shown that exposure to NMs may be associated with an increased risk of certain diseases, not only for aquatic organisms (Federici et al. 2007; Baun et al. 2008; Kasemets et al. 2009; Blinova et al. 2010) but also for humans (Pope et al. 2002; Oberdörster et al. 2005). The behavior of NPs in the environment, as well as their fate remain unclear, and it is well known that their state of aggregation and their consequent settlement to sediments depend on the hydrodynamic properties of freshwater systems (Gregory 2006), making prediction of these phenomena almost unreliable.
Correspondence: Dr Paride Mantecca, PhD, Dept. Environmental Science, Research Centre POLARIS, University of Milano-Bicocca, 1 piazza della Scienza, I-20126 Milan, Italy. E-mail:
[email protected] ISSN 1743-5390 print/ISSN 1743-5404 online 2012 Informa UK, Ltd. DOI: 10.3109/17435390.2011.579634
382
R. Bacchetta et al.
Despite this significant gap in our understanding, it is well known that aquatic environments are certainly at risk of exposure to these pollutants, as they represent a sink for most environmental contaminants (Scown et al. 2010). Thus, considering NPs as a new class of hazardous material, we aimed to study the effects of metal oxide-based NPs on the amphibian Xenopus laevis using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) test. The three endpoints of this test, i.e., mortality, malformations and growth inhibition, render it a powerful and flexible bioassay for evaluating pollutants in water (Dumont et al. 1983). FETAX can be used for testing single compounds (Bantle and Dawson 1988), as well as the joint actions of more than one compound (Dawson 1991). However, despite the great amount of literature available on the effects of many compounds on the larval development of X. laevis, only one report exists that deals with the teratogenic potential of a metal NP on this amphibian (Nations et al. 2011). This report paved the way for the present study, in which we investigated the effects of three different nano-sized metal-oxides (nCuO, nTiO2 and nZnO) on X. laevis embryos. Because the release of ions from metals may be a key factor in their toxicity (Kahru et al. 2008; Kasemets et al. 2009; Blinova et al. 2010), the effects of Cu++ and Zn++ were also evaluated. Luo et al. (1993) showed that these two metal ions were teratogens for X. laevis, and some authors (Franklin et al. 2007; Heinlaan et al. 2008; Aruoja et al. 2009; Blinova et al. 2010) have reported that in other animals, the toxicity associated with ZnO and CuO NPs was due to dissolved zinc and copper ions. In contrast, Griffitt et al. (2007, 2009) and Ispas et al. (2009) demonstrated that the effects of Cu and Ni NPs in fish are not simply mediated by dissolution, emphasizing the need for additional research on this topic. In a review entitled ‘From ecotoxicology to nanoecotoxicology’, Kahru and Dubourguier (2010) highlighted the most recent frontiers opened in the field of ecotoxicology by nanotechnology. This paper refers to the new scientific problems introduced by engineered nanoparticles (ENPs) in terms of environmental monitoring and modeling, as well as approaches that are useful to screen for their toxicity. These problems are mainly derived from the peculiar physico-chemical characteristics of the nanoworld, as well as from the unpredictable mechanisms of interaction between nanostructures and biological systems. Due to the complexity of phenomena at the nanolevel and the relatively young age of this discipline, no instruments or methods are currently available to provide standardized measures of
environmental ENP concentrations, or even their speciation. The urgent need for standardized approaches for NP hazard identification and evaluation prompted our use of X. laevis development as a ‘nanoecotoxicological model’ to screen the embryotoxic and teratogenic potential of NPs in a freshwater environment. Using histopathology and advanced confocal and transmission electron microscopy, we focus on the crucial topic of bridging the standard outputs of such toxicity tests with the mode of NP-tissue interaction and internalization.
Materials and methods Chemicals and NPs used All analytical grade reagents, human chorionic gonadotropin (HCG), 3-amino-benzoic acid ethyl ester (MS222), salts for FETAX solutions, CuSO45 H2O, ZnSO4 and metal oxide NPs were purchased from Sigma-Aldrich S.r.l., Italy. The advertised sizes of these particles were
