The exponential growth of nanotechnology has led to the production of large quantities of nanomaterials for numerous industrial, technological, agricultural, environmental, food and many other applications. However, this huge production has raised growing concerns about the adverse effects that the release of these nanomaterials may have on the environment and on living organisms. Regarding the effects of QDs on aquatic organisms, existing data is scarce and often contradictory. Thus, more information is needed to understand the mechanisms associated with the potential toxicity of these nanomaterials in the aquatic environment. The toxicity of QDs (ZnS and CdS) was evaluated in the freshwater fish Danio rerio. The fishes were exposed for seven days to different concentrations of QDs (10, 100 and 1000 µg/L) individually and combined. Oxidative stress enzymes (catalase, superoxide dismutase and glutathione S-transferase), lipid peroxidation, HSP70 and total ubiquitin were assessed. In general, results suggest low to moderate toxicity as shown by the increase in catalase activity and lipid peroxidation levels. The QDs (ZnS and CdS) appear to cause more adverse effects singly than when tested combined. However, LPO results suggest that exposure to CdS singly caused more oxidative stress in zebrafish than ZnS or when the two QDs were tested combined. Levels of Zn and Cd measured in fish tissues indicate that both elements were bioaccumulated by fish and the concentrations increased in tissues according to the concentrations tested. The increase in HSP70 measured in fish exposed to 100 µg ZnS-QDs/L may be associated with high levels of Zn determined in fish tissues. No significant changes were detected for total ubiquitin. More experiments should be performed to fully understand the effects of QDs exposure to aquatic biota.

Polycyclic Aromatic Hydrocarbons (PAH) are a class of organic pollutants normally found as mixtures with effects often hard to predict, which poses a major challenge for risk assessment. In this study, we address the effects of Phenanthrene (Phe), benzo[b]fluoranthene (B[b]F) and their mixtures (2 Phe:1 B[b]F; 1 Phe: 1 B[b]F; 1 Phe: 2 B[b]F) over glutathione (GSH) synthesis and function in HepG2 cells. We analyzed the effects on cellular viability, ROS production, glutathione (GSH) levels, protein-S-glutathionylation (PSSG), the activity of glutathione peroxidase (GPx), glutathione-S-transferases (GST) and glutathione reductase (GR). Transcript (mRNA) levels of glutathione synthesis enzymes - glutathione cysteine ligase catalytical (GCLC) and modifying (GCLM) sub-units and glutathione synthetase (GS) - and Nrf2 translocation to the nucleus were analyzed. Phe showed a higher cytotoxicity (IC₅₀ = 130 µM after 24 h) than B[b]F related to a higher ROS production (up-to 50% for Phe). In agreement, GSH levels were significantly increased (up-to 3-fold) by B[b]F and were accompanied by an increase in the levels of PSSG, which is a mechanism that protect proteins from oxidative damage. The upregulation of GSH was the consequence of Nrf2 signaling activation and increased levels of GCLC, GCLM and GS mRNA observed after exposure to B[b]F, but not during exposure to Phe. Most interestingly, all mixtures showed higher cytotoxicity than individual compounds, but intriguingly it was the 1 Phe: 1B[b]F mixture showing the highest cytotoxicity and ROS production. GSH levels were not significantly upregulated not even in the mixture enriched in B[b]F. These results point to the role of GSH as a central modulator of PAH toxicity and demonstrate the idiosyncratic behavior of PAH mixtures even when considering only two compounds in varying ratios.

Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 10⁷ cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.