Nanochemistry and biotechnology
Prooxidant-antioxidant status of rat hepatocytes mitochondria under incubation with GdVO4:Eu3+ nanoparticles in vitro

Kot Yu.1, Kot K.1, Andreev D.1, Klochkov V.2, Kavok N.2, Yefymova S.2, Malyukin Yu.2, Perskyi Ye.1 

1 V. N. Karazin Kharkiv National University, biochemistry department, 4 Svobody Sq., Kharkiv, 61022, Ukraine.
E-mail: kot.juriy@gmail.com
2 Institute for Scintillation Materials, Nanocrystal Materials Department, Nauky ave. 60, Kharkiv, 61072, Ukraine.

It was shown, that GdVO4:Eu3+ nanoparticles (D=3nm), which had been synthesized by the Nanocrystal Materials Department of Institute for Scintillation Materials of National Academy of Sciences of Ukraine, had a high redox activity and regenerative capacity. Therefore, GdVO4:Eu3+ nanoparticles (D=3nm) are perspective on using as a modulator of prooxidant-antioxidant processes in mammalian cells.
The aim of the research was to study the sorption dynamics of GdVO4:Eu3+ nanoparticles (D=3nm) into hepatocytes mitochondria and prooxidant-antioxidant status of hepatocytes mitochondria in vitro. The following indicators were measured: concentration of reactive oxygen species, lipid hydroperoxides and ATP, mitochondrial potential change, activity of superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase.
In our research, we used isolated hepatocytes of Wistar rats at the age of 3 months. Vitality of hepatocytes was 87% (Invitrogen Countess). Cells were incubated with nanoparticles at a concentration of 1.0 μg/104 cells within 10 min, 30 min, 90 min and 180 min. Then, live cells and mitochondria, which were obtained by differential centrifugation, washed off from unbound nanoparticles and analyzed by scanning fluorimeter Bio-Tek FL600 (Ex/Em=360/618 nm). The concentration of reactive oxygen species (DCFDA, Ex/Em=495/529 nm), lipid hydroperoxides (MitoPeDPP, Ex/Em=452/470 nm) and ATP concentration (LumiGlo) was measured. Also, the extent of mitochondrial potential change (JC-1, Ex/Em=485, 540/535, 570 nm) and enzymatic activity of superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase (Abcam) were determined.
As a result of the hepatocytes incubation with nanoparticles within 180 minutes, the cells gradually accumulated particles from the tenth minute and into the mitochondria from the sixtieth minute. The maximum sorption of nanoparticles into cells and mitochondria occurred at 90th minute, but at 180th minute, their sorption decreased by 10%. It was identified, that nanoparticles exhibit the characteristics of prooxidants. There was an increase in the concentration of ROS and lipid hydroperoxides in the mitochondria at 90th minute of incubation. At the same time, the concentration of ATP and the mitochondrial potential decreased. It can be assumed that nanoparticles with the direct impact on the mitochondrial membrane damage it, causing dysfunction of the mitochondrial pores.
It was established that nanoparticles activated the enzymes of the antioxidant system at 10th minute of incubation, long before the commencement of nanoparticles sorption into mitochondria. This prooxidant effect of nanoparticles on cells can be the result of ROS increasing in the cytoplasm, when they pass through the plasma membrane or could be a consequence of the nanoparticles ability to influence on the enzymes synthesis de novo.