engleski

Assessment of reactive oxygen species in tobacco (Nicotiana tabacum L.) seedlings exposed to silver nanoparticles

Silver nanoparticles (AgNPs) have wide application in many consumer products due to their unique chemical and physical features enhancing well known antibacterial and antifungal properties of silver. Increase in AgNPs production has raised many concerns about their possible toxicity, and many studies have already indicated that they induce oxidative stress through enhanced production of reactive oxygen species (ROS). Rapid accumulation of ROS and imbalance in their generation and scavenging could result in severe damage of proteins, lipids and DNA. Since many factors control the ROS metabolism, detecting ROS accurately is challenging. The aim of this study was to develop fast and reliable method for quantitative determination of ROS in the plant tissue and to test the method in AgNP-treated tobacco (Nicotiana tabacum) seedlings. Three weeks old seedlings were treated with 25, 50, 75, 100 and 150 μM polyvinylpyrrolidone (PVP)-coated AgNPs for 7 days. Cytosolic ROS level in the plant extracts was assessed with fluorescent probe dihydroethidium (DHE) that specifically detects superoxide radical, and for quantification of fluorescence, microplate reader was set at 520 nm for excitation and 600 nm for emission. To optimize the method, optimal incubation time of the reaction was tested by monitoring fluorescence immediately after addition of DHE, and after 5 and 15 minutes of incubation. The linearity of the method was tested by measuring florescence of several dilutions of the extracts. Testing the incubation time for the samples showed that incubation could lead to false results and should be omitted. Moreover, measuring several dilutions of the samples confirmed linearity of the method thus proving that this method could be used for ROS quantification in the plant extracts. Finally, obtained results showed dose-dependent increase of ROS in AgNP-PVP treated tobacco seedlings’ indicating that oxidative stress is involved in toxicity of AgNPs towards plants.

AgNP ; tobacco seedlings ; oxidative stress ; ROS

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