Catalytic activity and expression of isoforms of antioxidant enzymes in tobacco seedlings (nicotiana tabacum l.) After treatment with nanosilver particles

Nanočestice srebra (AgNP) zbog svojih specifičnih svojstava imaju široku upotrebu, no trenutno nema puno saznanja o njihovom potencijalno toksičnom utjecaju na okoliš i čovjeka. Cilj ovog istraživanja bio je ispitati uzrokuju li AgNP povećanu razinu oksidacijskog stresa, kao jednog od mogućih mehanizama njihove toksičnosti. Modelni organizam, klijanci duhana (Nicotiana tabacum L.) tretirani su AgNP stabiliziranim s omotačem od polivinilpirolidona (AgNP-PVP) u koncentracijama od 25, 50, 75, 100 i 150 μM sedam dana. Nakon tretmana u proteinskom ekstraktu praćena je aktivnost i ekspresija izoformi antioksidacijskih enzima: superoksid dismutaze (SOD), katalaze (CAT), askorbat peroksidaze (APX) i pirogalol peroksidaze (PPX). Katalitička aktivnost enzima SOD, CAT, APX i PPX određena je spektrofotometrijski, a njihove izoforme su analizirane nativnom elektroforezom na poliakrilamidnom gelu (PAGE). Dobiveni rezultati obrađeni su statistički (Duncan test, razina značajnosti p < 0,05). Tretman s AgNP-PVP nije imao značajniji utjecaj na aktivnost enzima SOD i CAT u klijanaca duhana, ali je uzrokovao značajno smanjenje aktivnosti APX i PPX u usporedbi s negativnom kontrolom (klijancima koji nisu bili izloženi AgNP-PVP). Također, nakon tretmana s AgNP-PVP u klijanaca duhana uočena je različita ekspresija izoformi PPX u odnosu na kontrolu. Povećana ekspresija izoformi PPX1 i PPX2 zabilježena je u klijanaca koji su bili izloženi 75, 100 i 150 μM AgNP-PVP, dok je najveća ekspresija izoformi PPX4 i PPX5 zabilježena u kontroli. AgNP-PVP nisu imale značajniji učinak na ekspresiju izoformi ostalih ispitivanih enzima. Dobiveni rezultati pokazuju da AgNP-PVP utječu na katalitičku aktivnosti i ekspresiju izoformi enzima koji su uključeni u antioksidacijsku obranu u biljci duhana. Stoga se može zaključiti da je oksidacijski stres uključen u mehanizam toksičnosti AgNP-PVP.Due to their specific properties silver nanoparticles (AgNP) have numerous applications. However, there is currently little knowledge about their potentially toxic effects on the environment and humans. The aim of this study was to examine whether AgNPs cause increased levels of oxidative stress, as one of the possible mechanisms of their toxicity. Model organism tobacco (Nicotiana tabacum L.) seedlings were treated with AgNP coated with polyvinylpyrrolidone (AgNP-PVP) at concentrations of 25, 50, 75, 100 and 150 μM for seven days. After the treatment, activity and expression of isoforms of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and pyrogallol peroxidase (PPX) in plant protein extracts were monitored. The catalytic activity of the enzymes SOD, CAT, APX and PPX was determined spectrophotometrically, and their isoforms were analyzed by native polyacrylamide gel electrophoresis (PAGE). The obtained results were statistically analysed (Duncan test, significance level p < 0.05). Treatment with AgNP-PVP had no significant effect on SOD and CAT enzyme activity in tobacco seedlings, however caused a significant reduction in APX and PPX activity compared to the negative control (seedlings that were not exposed to AgNP-PVP). Also, after treatment with AgNP-PVP in tobacco seedlings different expression of PPX isoforms was observed compared to the negative control. Increased expression of PPX1 and PPX2 isoforms was observed in tobacco seedling exposed to 75, 100, and 150 μM AgNP-PVP, while the highest expression of PPX4 and PPX5 isoforms was observed in the negative control. AgNP-PVP did not have a significant effect on the expression of isoforms of other investigated enzymes. The results of this study show that AgNP-PVP affects the catalytic activity and the expression of enzyme isoforms involved in antioxidant defense in the tobacco plant. Therefore, it can be concluded that oxidative stress is involved in the mechanism of AgNP-PVP toxicity