Falfushynska_2019_Saudi.J.Biol.Sci_26_481

Global decline of amphibian populations causes particular concern about their vulnerability to novel environmental pollutants, including engineering nanomaterials and pharmaceutical products. We evaluated the bioavailability of nanoform of zinc oxide (n-ZnO) in frog Pelophylax ridibundus and determined whether co-exposure to a common pharmaceutical, a calcium-channel blocker nifedipine (Nfd) can affect this bioavailability. Male frogs were exposed for 14 days to the tap water (Control) and n-ZnO (3.1 muM), Zn(2+) (3.1 muM, as a positive control for n-ZnO exposures), Nfd (10 muM), and combination of n-ZnO and Nfd (n-ZnO + Nfd) in environmentally-relevant concentration. Exposure to Zn(2+) or n-ZnO led to up-regulation of metal-binding proteins, metallothioneins (MTs) in the liver and Zn-carrying vitellogenin-like proteins in the blood plasma. Notably, upregulation of MTs by Zn(2+) or n-ZnO exposures combined with increased binding of Zn and Cu to MTs. This was associated with the more reducing conditions in the liver tissue indicated by elevated lactate to pyruvate ratio. Nfd suppressed the binding of Zn and Cu to MTs and led to a decrease in Lactate/Pyruvate ratio and elevated protein carbonylation indicating pro-oxidant conditions. Redox status parameters were not directly related to DNA fragmentation, nuclear abnormalities or suppression of cholinesterase activity indicating that factors other than oxidative stress are involved in cytotoxicity of different pollutants and their combinations. Furthermore, activity of Phase I biotransformation enzyme (CYP450 oxidase measured as EROD) was elevated in Nfd-containing exposures and in Zn(2+) exposed frogs. Tyrosinase-like activity in the frog liver was strongly stimulated by Zn(2+) but suppressed by n-ZnO, Nfd and n-ZnO + Nfd. These findings show that Nfd modulates homeostasis of essential metals in amphibians and emphasize that physiological consequences of combined n-ZnO and Nfd exposures are difficult to predict based on the mechanisms of single stressors.