Bioaccumulation, subcellular partitioning and biomarkers at different levels of biological organization of aquatic invertebrates exposed to metallic nanoparticles

Abstract

The study aims to elucidate the bioavailability/bioaccumulation, mechanisms of action and biological damage related to exposure of aquatic organisms to metals and metal nanoparticles (NP) that are fundamental for the evaluation of the environmental impact of atmospheric particulate matter (MPA) in continental and marine ecosystems and answer the following questions: (i) What is the effective bioavailability of metals and metallic NP in aquatic organisms? (ii) Once absorbed, which cellular tissues and organelles have been impacted? (iii) What genetic, biochemical and morphological responses are induced by exposure to MPA? (iv) From what concentration do NP induce adverse effects? The study will be developed with MPA samples collected near the Tubarão Complex, Espírito Santo, Brazil, in the aquatic biotery of the Institute of the Sea (DCM) of the Federal University of São Paulo (UNIFESP), Campus Baixada Santista (Santos, SP) using the crustacean, Litopenaeus vannamei and the mollusk, Perna perna and fish, Centropomus parallelus. The animals will be exposed to at least 2 concentrations of MPA (MPA groups) and the respective control groups (groups C) will be kept in water without adding MPA. The duration of exposure will be determined after the evaluation of the bioaccumulation dynamics of metals and NPs present in MPA. After exposure, samples of hemolymph, gills, hepatopancreas or digestive gland, gonads and muscle will be removed and stored at -80 °C for the analyses that will be performed later: quantification of metals and NPs in tissues and subcellular partition, internalization of NPs, genotoxic, molecular and biochemical analyses. The determination of the dynamics of total bioaccumulation, differential bioaccumulation in organs and tissues will be performed after lyophilization and acid digestion of the samples and multielementary analysis that will be performed in ICP-MS. For subcellular analyses, homogenized tissues will be subjected to differential centrifugations using temperature and acid digestion procedures. Metals will be quantified in mitochondria (P3), lysosomes and microsomes (P4), enzymes and heat-sensitive proteins (P5). The presence of metals in these subcellular fractionations will be grouped together into Biologically Active Metals (MBA). While metals quantified in granules (P2) and final supernatant, containing heat-stable protein and metalotionein protein (S5) will be grouped as Biologically Detoxified Metals (MBD). Metals will be dosed in ICP-MS in the following subcellular fractionations: mitochondria (P3), lysosomes and microsomes (P4), heat-sensitive proteins, enzymes (P5) (MBA), granules (P2), heat-stable protein and metallothionein protein (S5) (MBD), nuclei and cellular debris (S2). Genotoxic and mutagenic evaluations will be carried out using the Comet and Micronucleus test and molecular/biochemical responses will be evaluated by the production of reactive species of O2 (EROs), activity of biotransformation enzymes (phase I, EROD and phase II, glutathione S-transferase, GST), antioxidant defenses (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx), levels of glutathione (GSH) and metalothionein (MT) and oxidative stress biomarkers: lipid peroxidation (LPO) and carbonylated proteins (PCO).