Effect Of Diclofenac On Respiratory Metabolism And Mitochondrial Function Of Tobacco BY-2 Cells

With the rapid development of economy and the improvement of human medical and health conditions, more than 3000 kinds of drugs have been used in medical care, and many of them accumulate in the environment. In fact, some pharmaceuticals are not totally eliminated because the conventional technology of treatment used in sewage treatment, plants appear to insufficiently remove these specific compounds. Consequently, discharged pharmaceuticals in variable quantities can reach surface waters, ground waters and sediments,with global universality in the environment. Among them, diclofenac is one kind of Nonsteroidal anti-inflammatory drugs(NSAIDs) that is frequently used in the treatment of inflammation and pain. Because of the curative effect and the large dosage, diclofenac is continuously released into the environment, endangering the stability of ecosystem.It has been known that diclofenac, the non-steroidal anti-inflammatory drug, widely used in clinical therapeutics, influences the growth and development of plants seriously.However, it has not been elucidated the mechanism by which the diclofenac inhibits plant growth. The tobacco BY-2 cells were used in this study to explore the inhibition mechanism.In this study, we explored the effect of diclofenac on respiratory metabolism and mitochondrial function of tobacco BY-2 cells. We used cells dry weight to reflect cell growth,which intuitively reflected inhibition of diclofenac on cells growth. The effects of diclofenac in different concentrations and in different treatmeat time on total respiration and via COX and via AOX pathway capacity in tobacco BY-2 cells and the cells respiration treated with respiratory pathways inhibitors and the instant inhibition effect of diclofenac on different respiratory states of tobacco BY-2 cells mitochondria were examined by measuring the oxygen consumption in tobacco BY-2 cells with an Oxytherm oxygen electrode(Hansatech,UK) to elucidate the mechanism of diclofenac inhibiting the growth of the tobacco BY-2 cells and to provide a theoretical basis for understanding the inhibiting mechanism of growth of plants by diclofenac.The results showed that after treated with diclofenac for 24 h, the growth of tobacco BY-2 cell was significantly inhibited, a burst and accumulation of reactive oxygen species(ROS)in the tobacco BY-2 cells were induced, and diclofenac promoted death of the tobacco BY-2cells. The results demonstrated that diclofenac has instant inhibition effect on respiratory oftobacco BY-2 cells depending on the drug concentration. The instant inhibition effect of 0.2mmol·L-1diclofenac was the most significant.In the study of the direct effect of diclofenac to isolated mitochondria, we found that by inhibiting cell mitochondrial respiratory electron transport chain complex II, III and IV activities, diclofenac resulted in the respiratory electron transport via COX and AOX were inhibited in different degrees, and then feedback inhibited the glycolysis(EMP), tricarboxylic acid cycle(TCA) and the pentose phosphate pathway(PPP), three carbon metabolic pathways,in tobacco BY-2 cells. Diclofenac inhibited state-I, state-III, and state-IV respiration rate, and lowered respiratory control rate(RCR). Furthermore, diclofenac induced mitochondrial swelling, and reduced the aggregation degree of mitochondrial membrane. Thus diclofenac undermined the integrity of the mitochondria, which hindered the formation of the transmembrane proton gradient and inhibited the synthesis of ATP. Therefore, we considered that the electron transport through complex III and IV of mitochondrial respiratory electron transport chain inhibited was the main reason of diclofenac inhibiting cells respiration.The study proved that inhibiting the activity of the complex II, III and IV in respiratory electron transport chain is the main reason to restrain cells respiration and to disturb mitochondrial metabolism. The disorders in cellular material metabolism and energy metabolism the burst and accumulation of reactive oxygen species induced by the diclofenac were the main reasons for diclofenac to inhibit growth of tobacco BY-2 cells and to lead to cells death.