Oxidative stress and genotoxicity in Rhinella arenarum (Anura: Bufonidae) tadpoles after acute exposure to Ni-Al nanoceramics
Graphical abstract
Introduction
The increased employ of nanomaterials (NMs) in several society areas will inevitably cause their release into the environment, especially into the aquatic matrix, during their production, transport, use and disposal (Mueller and Nowack, 2008). Moreover, NMs can accumulate in sediments or remain suspended, and aquatic organisms can interact with them (Rocha et al., 2015).
Alumina (Al) is one of the most widely used oxide ceramic material, and it is estimated to represent approximately 20 % of the 2013 worldwide marketplace of nanoparticles (NPs) (Future Markets Inc., 2013). <gamma>-Al based NMs are used in several commercial products such as seal rings, medical prostheses, electronic substrates, ballistic armour, thermocouple tubes, electrical insulators and also in wear components. It is interesting to underline that < gamma>-Al2O3 has significant applications as nanomembranes with nanometric size pores and it is highly used as catalytic support due to its extensive surface area, thermal stability and low cost (Rozita et al., 2010). That is the case of the industry of methane reform that uses nickel supported on alumina (Ni/Al2O3) as a catalyst. Due to its intrinsic characteristics as small size and high surface area, NMs can cause high toxicity. Therefore, it is necessary to evaluate its impact both on environment and health (Handy et al., 2008; Exbrayat et al., 2015; Ferreira do Amaral et al., 2019).
Amphibians are frequently employed in the ecotoxicity field as a representative aquatic group and are considered valuable indicators of changes and stressors in polluted areas (Burlibaşa and Gavrilacaron, 2011). Among them, the anuran Rhinella arenarum is considered a good sentinel organism due to its high sensitivity because of their biphasic life cycle, permeable eggs, skin and gills. Moreover as they live in or near water reservoirs they are directly or indirectly affected by aquatic pollutants. Tadpoles are filter-feeders, so pollution can therefore happen via skin contact and water filtration, but tadpoles can also graze the NPs at the surface of sediment. Some authors have already evaluated the toxicity of other NPs on this same species (Yslas et al., 2012; Ibarra et al., 2015, 2016). In our previous studies we evaluated lethality (Svartz et al., 2017) and sublethal effects as neurotoxicity, morphological and histological changes of Ni-Al nanoceramics on embryos and larvae of this species (Svartz et al., 2019). Subcellular biomarkers of oxidative stress and genotoxicity are considered efficient tools to evaluate early signs of pollutant exposure. These biomarkers have higher sensitivity than physiological and morphological changes because they depend on the chemical mechanism of action (Jemec et al., 2008). Many pollutants can inflict toxic effects by the generation of reactive oxygen species (ROS), specially the NMs because of several properties as size, surface area, and surface chemistry (reactive groups) (Ferreira do Amaral et al., 2019). Organisms have antioxidant defenses against ROS, which include molecules such as reduced glutathione (GSH), which is a valuable biomarker of oxidative stress in amphibians (Ferrari et al., 2009), and a battery of antioxidant enzymes including, e.g., superoxide dismutase (SOD) and catalase (CAT) (Halliwell and Gutteridge, 2015). Particularly, these two enzymes have shown high levels of activity throughout the first stages of amphibian development. GSH content is also considered a valuable biomarker of oxidative stress in amphibians (Ferrari et al., 2009). Glutathione S-transferase (GST) is another important enzyme which also plays an antioxidant role (Halliwell and Gutteridge, 2015). However, an excess of ROS can alter membranes, biological macromolecules, ion leakage, and lead to lipid peroxidation and DNA-strand cleavages (Valavanidis et al., 2006; Khanna et al., 2015). DNA damage is often measured by the analysis of micronuclei (MNs), small fragments of intracytoplasmic chromatin, caused by chromosomal breaks or whole chromosomes (Gauthier et al., 1993).
The synthesis of Ni/<gamma>-alumina catalytic nanoceramic involves NMs: Ni/<gamma>-Al2O3 (NC), <gamma>-Al2O3 support (SPC) and NiO/<gamma>-Al2O3 precursor (PC). In previous works we have reported lethality and morphological alterations of these NMs in R. arenarum early development. In this study, we have focused on both oxidative stress damage and the genotoxic potential of NC, PC and SPC on the enzymatic and non-enzymatic antioxidant system of R. arenarum larvae. Biomarkers of oxidative stress (CAT, SOD, GST, GSH and lipid peroxidation) and genotoxic damage through MNs frequencies in peripheral blood erythrocytes were analyzed. The main hypotheses of this study is that NMs disrupt the antioxidant system causing oxidative stress damage (lipid peroxidation) and genotoxic effects in R. arenarum larvae, with differential responses between NMs related to reactivity characteristics. It is important to highlight the importance of the study of sublethal effects. On one hand, the exposure to low concentrations of a given pollutant represents the most common environmental scenario and on the other hand, they could generate an indirect risk by means a reduction in fitness, which may provoke a long-term decrease in the exposed population and consequent loss of biodiversity (Saaristo et al., 2018).
Section snippets
Nanoparticles
Three types of Al nanoceramics were obtained from sol-gel method according to procedures by Perez-Catán and Guraya (2015): <gamma>-Al2O3 (SPC), NiO/<gamma>-Al2O3 (PC) and Ni/<gamma>-Al2O3 (NC). The synthesis involved the hydrolysis of aluminium sec-butoxide, followed by gelation process that become in a porous solid by thermic treatment. The structure of the solid was characterized by means Nitrogen Adsorption/Desorption Isotherms at 77° K, in a Digisorb 2600 from Micromeritics Int. Corp. The
Characterization of NP suspensions
Table 1 shows the physicochemical characteristics of the NPs. The specific surface area of SPC, PC and NC diminished according to nickel chemical species (NiO or Ni). NC exhibited the lowest surface area. These differences could affect the reactivity of NPs. All parameters of porous structure were higher in SPC and decreased in PC and NC. The average particle size was below 50 nm and the x-ray spectra showed a matrix structure with Nickel NPs forming primarily hexahedral-type crystals and γ-Al2O
Discussion
The increased production and use of NMs, due to their wide applications and unique properties, resulted in the release of huge quantities of these substances into the environment without knowledge of their effects. Therefore, it is essential to evaluate the negative impacts of these NPs on aquatic biota and to study the toxicity mechanisms. In the present study, we analyzed the alterations of enzymatic and non-enzymatic defense system, oxidative stress damage and genotoxic effects of a
Conclusions
The results obtained in this study clearly reveal that oxidative stress and genotoxicity are the main toxicity factors of these NMs on R. arenarum larvae and the possible responsible mechanisms for the sublethal effects previously reported that may affect the fitness and performance of exposed populations. These subcellular biomarkers could be very useful tools as early warning indicators of the potential long-term effects of these NMs on R. arenarum populations.
CRediT authorship contribution statement
Gabriela Svartz: Investigation, Methodology, Formal analysis, Data curation, Software, Writing - original draft, Writing - review & editing. Carolina Aronzon: Formal analysis, Writing - review & editing. Soledad Pérez Catán: Methodology, Resources. Sonia Soloneski: Methodology, Visualization, Writing - review & editing. Cristina Pérez Coll: Conceptualization, Writing - review & editing, Visualization, Supervision, Validation, Resources, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors thank Ferring Pharmaceuticals for providing human chorionic gonadotropin hormones. This study was partially supported by MINCYT–PICT2017-0706. This study was supported by a grant from the National Agency of Scientific and Technological Promotion (PICT 2017 Number 0706) from Argentina.
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