The widespread use of herbicides in agriculture leads to their accumulation in the soil, which is dangerous not only for plants, but also for soil microorganisms, animals and humans. At the same time, the effect of many commonly used herbicides on cell metabolism is still not well understood. In this work, we studied the cytotoxic effect of the herbicides fluorodifen and diuron on heterotrophic plant cells. It is known that the negative effect of these chemical agents on weeds is mainly due to inhibition of photosynthesis and the associated oxidative stress however heterotrophic plant cells lack the main targets for their action. A 7-day suspension culture of Arabidopsis thaliana L. cells was chosen as the object of study. The cell culture was grown in the dark at 26 °C in flasks (250 ml) with constant stirring in Murashige-Skoog medium containing sucrose (36 g/L), nicotinic acid (0.6 mg/L), pyridoxine (0.6 mg/L), thiamine (1.2 mg/ L), 2,4-D (3 mg/L), inositol (120 mg/L), and sodium dithiocarbamate (6 mg/L). The cell culture was transplanted every two weeks with a new medium dilution of 6 times and used for experiments on the 7th day after transplantation. Arabidopsis suspension culture cells were treated with herbicides of various concentrations: diuron – 50, 100, 200 μM; fluorodifene – 10, 100, 200 μM. To assess the effect of herbicides on cell metabolism, fluorescent probes were used: FDA (fluorescein diacetate), PI (propidium iodide), H₂DCF-DA (2’, 7’-dichlorofluorescein diacetate) and JC-1 (5,5',6,6'-tetrachloro-1,1',2,2'-tetraethylbenzimidazolocarbocyanine). An Axio Observer Z1 inverted fluorescence microscope (Carl Zeiss Microscopy, Germany) with an AxioCam MRm3 digital monochrome camera and an AxioVision Rel.4.7 image analysis software package were used. At the first stage, the phytotoxic effect of various concentrations of diuron and fluorodifene was analyzed, treating the cells for 24 hours at 26 °C. At the control condition, the number of living cells (cells that were stained with FDA and not stained with PI) ranged from 70 to 80 %. Diuron caused a significant decrease in cell viability only in the case of incubation of cells with the maximum used concentration (200 μM). The proportion of living cells in the culture in that condition was 30 %. Fluorodifene at a concentration of 10 μM did not cause the death of Arabidopsis cells, however, an increase in its concentration to 100 and 200 μM led to death to 35 and 45 % of the cells, respectively. Thus, the concentrations of herbicides that caused an approximately twofold decrease in viability within 24 h as compared with the control were determined: for diuron and fluorodifene they amounted to 200 μM. It should be noted the toxic effect of these herbicides manifested itself after 1 h of treatment. In this case, the death of arabidopsis cells under the influence of diuron was accompanied by an increase in the reactive oxygen species (ROS) level and a short-term increase in the electrochemical transmembrane potential on the inner mitochondrial membrane (ΔμН⁺) in the cells. At the same time, the toxic effect of fluorodifen was excellent – cell death in the culture was not accompanied by changes in the levels of ROS and ΔμH⁺. The results obtained indicate that the cytotoxic effect of herbicides may not be associated with the development of oxidative stress in cells and depends on the characteristics of the metabolism of the target cell.

Fedyaeva Anna Valeryevna, Candidate of Science (Biology), Research Scientist, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation, e-mail: fedyaeva.anna@mail.ru 

Li I, Postgraduate, Irkutsk State Agrarian University named after A.A. Ezhevsky, Molodezhny settl., Irkutsk Region, 664038, Russian Federation, e-mail: li05161020@163.com 

Lyubushkina Irina Viktorovna, Candidate of Science (Biology), Research Scientist, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation; Assistant Professor, Irkutsk State University, 1, Lenin st., Irkutsk, 664033, Russian Federation, e-mail: ostrov1873@yandex.ru 

Fedoseeva Irina Vladimirovna, Candidate of Science (Biology), Senior Research Scientist, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation, e-mail: fedoseeva@sifibr.irk.ru 

Sidorov Aleksandr Vladimirovich, Lead Engineer, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation; Assistant, Irkutsk State Medical University, 1, Krasnogo Vosstaniya st., Irkutsk, 664033, Russian Federation, e-mail: a_v_sidorov@mail.ru 

Rikhvanov Evgenij Gennad'evich, Doctor of Sciences (Biology), Principal Research Scientist, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation, e-mail: eugene@sifibr.irk.ru

Fedyaeva A.V., Li I., Lyubushkina I.V., Fedoseeva I.V., Sidorov A.V., Rikhvanov E.G. Study of the Cytotoxicity of the Herbicides of Diuron and Fluorodifen using a Heterotrophic Suspension Cell Culture of Arabidopsis thaliana L. The Bulletin of Irkutsk State University. Series Biology. Ecology, 2019, vol. 30, pp. 16-31. https://doi.org/10.26516/2073-3372.2019.30.16 (in Russian)

Arabidopsis thaliana L., suspension cell culture, herbicides, diuron, fluorodifen, reactive oxygen species, electrochemical transmembrane mitochondrial potential

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