Tissue-specific genotoxicity and antioxidant imbalance of titanium dioxide nanoparticles (NPTiO2) and inorganic lead (PbII) in a neotropical fish species
Introduction
Nanomaterials belong to the class of emerging contaminants and they are defined as materials, which structure have between 1 and 100 nm in at least one of their dimension (Sauvé and Desrosiers, 2014; Snow et al., 2019). By having unique characteristics, such as large surface area, high reactivity and penetration ability, they are very attractive to industrial and medical technologies (Oberdörster et al., 2005; Borm et al., 2006; Kreyling et al., 2006; Lam et al., 2006; Maynard, 2007). One of the synthetic nanomaterials that have dramatically increased production is the titanium dioxide nanoparticle (NPTiO2) (USEPA, 2014; Miao et al., 2015). In nanometric scale, titanium dioxide has a higher refractive index, and for this reason, it is widely used in the production of sunscreens, cosmetics, and textiles.
Due to the many advantageous physicochemical characteristics for commercial production (e g. chemical reactivity and physical absorption), nanoparticles are also of concern because once they reach the aquatic environment they are able to interact with the most diverse molecules present there (Farré et al., 2009). The main exposure routes described for fish to metallic nanoparticles are via ingestion (both water and contaminated food), dermal (body epithelium) or breathing (gill epithelium and olfactory organs) (Moore, 2006). Since NPTiO2 is internalized by organisms, at the cellular level, the damage found ranges from the generation of reactive oxygen species (ROS) in excess, to DNA damage via oxidative stress, until the direct interaction of nanoparticles with proteins involved in the cell cycle (Warheit et al., 2007; Rahman et al., 2009). More recently, the description of a new mechanism of direct interaction of NPTiO2 with DNA in fish cells (gonadal lineage RTG-2) causing DNA damage has also been reported (Klingelfus et al., 2019).
The methods for quantifying nanoparticles in different environmental compartments (wastewater, plant effluents, biosolids, sediments, soils, and air) are yet to be optimized (Gottschalk and Nowack, 2011; Ma et al., 2016; Snow et al., 2019). It is a challenge to determine the concentration of nanoparticles present in these compartments. In rivers, for example, titanium dioxide nanoparticle concentrations estimated in modeling studies range from 3 ng L−1 to 1.6 μg L−1 (Gottschalk et al., 2013). It is important to be aware that bioassays conducted in different laboratory conditions may not expose fish species to representative concentrations found in the environment. It is noteworthy that a set of factors may influence the NPTiO2 biodistritution as these particles reach the aquatic environment (Farré et al., 2009). Considering this information, the direct administration of nanoparticles provides a more organism-specific response. In an attempt to generate a consistent database that will allow us to better understand the effects caused by exposure to these nanoparticles, some studies have also evaluated the toxic effects of NPTiO2 in fish through intraperitoneal (i.p.) injections (Vignardi et al., 2015; Jovanovic et al., 2015; Delmond et al., 2019).
Due to the increase in NPTiO2 production, there is a possible risk associated with the presence of metals, such as lead in aquatic environment. Lead (Pb) is one of the metals with high toxicity, persistence and bioaccumulative effects that can cause harmful damages on living organisms (Pain et al., 1995; Check and Marteel-Parrish, 2013; Wani et al., 2015).
In ecotoxicological studies, Pb induced responses such as abnormal swimming, skeletal deformities, enzyme inhibition, mutations, chromosomal aberrations, cancer and birth defects (Martinez et al., 2004; Cestari et al., 2004; Ferraro et al., 2004; Ramsdorf et al., 2009; Dos Santos et al., 2016; Reynolds et al., 2018). It is noteworthy that Pb affects the antioxidant defense system to induce generation of free radicals and reactive oxygen species (ROS) (Carocci et al., 2016). Some studies also suggest Pb act as a co-carcinogen, due to interference with DNA repair processes, thus enhancing the genotoxic effect of other agents, such as UV radiation (Fracasso et al., 2002).
There is evidence that metal ions can be adsorbed due to surface adsorption capacity of some metal nanoparticles (Sun et al., 2007; Zhang et al., 2007; Handy et al., 2008). However, studies that evaluate the co-exposure of nanoparticles with other compound are scarce (Sun et al., 2007; Zhang et al., 2007; Rossi et al., 2014; Vicari et al., 2018; Delmond et al., 2019) possibly due to the technical difficulties of conducting these experiments, such as the large surface area, crystalline structure, and reactivity of some nanoparticles, as well as characterization of the behavior of both substances together.
Toxicity studies with endemic species of tropical areas are of major importance, since much of the available data was generated with water quality parameters and species of temperate climate (Kwok et al., 2007; Rico et al., 2011). The species Rhamdia quelen is a omnivorous fish species with Neotropical distribution (Barcellos et al., 2004) and it has characteristics such as rapid growth, with easy adaptation to handling, good productivity in reservoirs and with high marketing potential (Gomes et al., 2000), besides being an important source of protein for population. This species is a good model for ecotoxicology studies (Pretto et al., 2010; Ferreira et al., 2010; Menezes et al., 2011; Mela et al., 2013; Ghelfi et al., 2016; Murussi et al., 2015; Golombieski et al., 2016; Pereira et al., 2016; Guiloski et al., 2017; Mathias et al., 2018).
The release of NPTiO2 and Pb into the aquatic environment is a reality, due to the continuous growth of production and the widely use of both in anthropic activities. Toxicity data based on the association of these substances are important to promote the development of effective regulatory policies. The aim of this study was to evaluate the genotoxicity and antioxidant system of the NPTiO2 alone and of its interaction with inorganic lead (Pb II) in fish blood, as well as in kidney and liver tissue of the neotropical fish Rhamdia quelen.
Section snippets
Fish
Specimens of Rhamdia quelen (Quoy et Gaimard, 1824) (Fish: HEPTAPTERIDAE) were purchased from a fish farm, located in Toledo (Paraná, Brazil). Fish were acclimated for 60 days in 2000 L tanks, with controlled temperature (28 °C) and photoperiod (12 h light/12 h dark), pH 7.0, constant aeration and water circulation. Additionally, they were feeding with commercial food once/day ad libitum (Primor, Brazil, 32 % protein).
Titanium dioxide nanoparticles suspensions - NPTiO2
NPTiO2 was purchased from Sigma-Aldrich® (Titanium (IV) oxide nanopowder, 21
Characterization of NPTiO2 suspensions
The transmission electron microscopy (Fig. 1) and the X-ray diffraction confirmed that the nanoparticle powder used is composed of 100 % anatase crystalline form. The material also showed a specific surface area of 83.47 m2 g−1, with an average size of 107 nm, 45 nm of dispersion size and surface chemistry comprising 28.42 % of titanium and 71.58 % oxygen.
The three NPTiO2 suspensions 0.05 mg L−1, 0.5 mg L−1 and 5 mg L−1 had an average particle size of 570.3 nm, 334.1 nm and two sizes for 5 mg L
Discussion
This study evaluated the potential toxicity of NPTiO2 and its co-exposure to the inorganic metal Pb utilizing intraperitoneal injections in the catfish Rhamdia quelen. Although intraperitoneal injections are not a way of exposure similar to which occurs in the aquatic environment, this model was chosen to allow the administration of an accurate dosage and consequently the possible interaction among the substances with known administered dosage.
Nanoparticles toxicity should not be limited to the
Conclusion
The NPTiO2 alone caused genotoxic effects in blood and kidney cells as well as the co-exposure with Pb, with the exception of the group exposed to 50 NP, which did not show genotoxic damage in the blood. Despite not being considered as a target organ, the effect observed in the kidney tissue must be considered carefully and, in the long-term exposure, the possibility of impairment of the functions of the kidney must be evaluated. The liver did not show genotoxic damage due to the NPTiO2
CRediT authorship contribution statement
Laís Fernanda Oya-Silva: Investigation, Methodology, Formal analysis, Visualization, Writing - original draft. Taynah Vicari: Supervision, Formal analysis, Writing - original draft, Writing - review & editing. Geonildo Rodrigo Disner: Methodology, Investigation. Juliana Roratto Lirola: Methodology, Investigation. Tatiane Klingelfus: Methodology, Investigation. Helyandra de Lourdes Schicora Gonçalves: Methodology, Investigation. Thalita Pires Borges Leite: Methodology, Investigation, Formal
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank Professor Wido Herwig Schreiner (Department of Physics - UFPR) and Professor Aldo José Gorgatti Zarbin (Department of Chemistry - UFPR), as well as LACTEC for helping with the characterization procedures of nanoparticles. This work is part of L. F. Oya Silva Master’s degree dissertation and was supported by CAPES(Brazilian National Higher Education Coordinating Council - Finance code 001). Additionally, the authors thank CNPq (Brazilian Council for Scientific and Technological
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These two authors contributed equally to this research.