| BackgroundDiquat(1,1'-ethylidene-2,2'-Bipyridine dibromide)is a kind of rapidly effective and non-selective herbicide and plant dehydrant,which belongs to the same class as the widely applicated paraquat(1-1-dimethyl-4-4 Bipyridine cation salt).Since it was first exploited and applicated in England in 1957,Diquat has been widely used all over the world because of its low cost,rapid onset and low pollution.With the ban on sales of paraquat in China in recent years,sales of Diquat soared,leading to the significant increase of patients with Diquat poisoning.However,at present,there is still no statistical data on the incidence of Diquat poisoning in China.Moreover,there is a high mortality rate of patients with Diquat,though Diquat is less toxic than paraquat.Previous studies found that Diquat can cause damage to kidney,lung,heart and so on,especially in the early stage,it can cause acute and severe renal failure.And severe poisoning can lead to multi-organ failure.The mechanisms of Diquat poisoning are complex and are not clear,and there is no specific antidote to Diquat at present.Therefore,it is of great significance to strengthen the basic and clinical research on Diquat poisoning.Objective1.To calculate the median lethal dose(LD50)of Diquat in rats by oral and observe the pathological changes of tissues and organs in rats with different concentrations of Diquat;2.To establish the rat model of acute Diquat poisoning and investigate the relationship between the concentration of Diquat in kidney and lung organs injury,in order to provide experimental basis for clinical treatment.Methods1.Detection of half lethal dose of Diquat to rats:Diquat solution of 50 mg-ml-1 was prepared freshly with 1000 mg of Diquat and dilute the solution with water to a total of 20 ml.A total of 99 healthy adult male Wistar rats were randomly divided into part one,part two and control groups.In the first part,36 rats were randomly divided into 4 groups:100 mg·kg-1 group,200 mg· kg-1 group,300 mg· kg-1 group and 400 mg· kg-1 group,which were treated with 100 mg· kg-1 200 mg· kg-1,300 mg· kg-1 and 400 mg· kg-1 of Diquat solution by gavage,respectively.The death and symptoms of poisoning after intragastric administration were recorded,and the maximum tolerated dose and absolute lethal dose were measured.In the second part,54 rats were randomly divided into 6 groups:200 mg· kg-1 group,220 mg· kg-1 group,240 mg· kg-1 group,260 mg· kg-1?280 mg· kg-1 group and 300 mg· kg-1 group,whichwere treated with 200 mg· kg-1,220 mg· kg-1,240 mg· kg-1,260 mg· kg-1,280 mg· kg-1 and 300 mg· kg-1 of Diquat solution by gavage,respectively.The survival of rats in different concentration of Diquat was observed and the LD50 was calculated by Excel processing the formula of Koch's method.The control group were given equal volume water under the same experimental conditions.And moreover,the lungs,kidneys,hearts,livers,and brain tissues were collected and fixed by formaldehyde,embedded by paraffin,and sectioned for histopathological light microscopy.2.Acute toxicity experiment:A total of 140 healthy adult male Wistar rats were randomly divided into the control groups and toxic groups.The intragastric concentration of the Diquat solution in the toxic groups were determined by the 1/2 of the lethal dose concentration.The control groups were given equal volume water.The dynamic changes of Diquat in plasma and tissues were quantitatively determined by the liquid chromatograph mass spectrometry at 0.5 h,1 h,2 h,4 h,8 h,16 h,24 h after poisoning.And at every time point,the content of hydroxyproline in lung tissues,the expression levels of serum BUN,creatinine,uric acid,the biological marker of early renal injury Kim-1 and TGF?1 were detected by western blot,and morphology of lungs and kidneys were measured.Results1.The LD50 of Diquat for rats by oral was 280.5837 mg·kg-1.And compared with the group with low-dose Diquat,the tissues and organs damages were obviously severe.The lungs were mainly infiltrated with inflammatory cells,the alveolar septum was thickened,and few fibril cords were formed.In renal tissues,the renal tubular epithelial cells were edematous and degenerated,glomerular capillaries were dilated,and endothelial cells were swelling.In liver tissues,the central hepatic veins were expansive,liver cells were swollen and structurally disordered.And moreover,myocardial and brain cells were swollen.As for dead rats,there was congestion in the alveolar space,increased infiltration of inflammatory cells,destruction of alveolar structures,and less formation of fibril cords;renal tubules and glomeruli hyperemia and edema,partial glomerular structure destruction,and structural disorder of myocardial fibers.2.The plasma concentrations of Diquat increased significantly at 0.5 h,1 h,2 h(P<0.01)after infection were(40.14±6.86)?(79.91±26.71)?(113.20±26.72)ng/ml respectively.The plasma concentrations of Diquat reached its peak at 2 h,then decreased,and the concentration decreased significantly at 4 h,while increased slightly at 8 h and 16 h,which was speculated to be related to the enterohepatic circulation.The concentrations of Diquat in kidney increased significantly at 1 h,2 h,4 h(P<0.01)after infection were(1575.70±86.68)?(2940.38±151.49)?(1775.69±49.73)ng/g respectively.The renal concentration of Diquat reached its peak at 2 h,and then decreased,while the concentration at 4 h was still higher,indicating that it was related to renal excretion,and then gradually decreased.The concentrations of Diquat in lung tissue increased significantly at 0.5 h,1 h,2 h after infection were(1.97±0.61)?(4.11±0.96)?(2.35±0.58)ng/g respectively.The concentration of Diquat in lung tissues peaked at 2 h,and then decreased to 24 h.Although the concentration of hydroxyproline in lung tissue was increased,it was not significant(P>0.05).Over time,the concentrations of serum uric acid,BUN,creatinine and Kim-1 were all increased with statistically significant(P<0.05),but the injury was slightly alleviate in the 4 h group after exposure,which was presumably related to the acute compensation of the body.Serum kim-1 in rats increased at 0.5 h and increased significantly at 4 h,which was an effective predictor of early renal injury.TGF?1 showed obvious early expression in renal tissue and no obvious expression in lung tissue.Electron microscopy showed that the degree of renal damage gradually increased with time.Vacuolation gradually increased,some mitochondrial bilayer membrane structures disappeared and lysosomes increased.The lung tissue damage was mild,and the cell membrane integrity and organelle were damaged to varying degrees.Conclusion1.The LD50 of Diquat for rats by oral was 280.5837 mg·kg-1.2.Diquat can cause damages of multiple organs in rats,with the higher the dose,the more serious the organ damage.3.In acute Diquat poisoning rats models,the concentration of Diquat in serum and tissues reached its peak at the early stage,and then decreased rapidly.4.The renal function damage and pathological changes were still persistent,while the lung tissue has mild injury accompanied by inflammatory cell infiltration and the early damage of pulmonary fibrosis was not obvious. |
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