Toxicity And Single-dose Toxicokinetics Study Of Diquat In Wistar Rats

Diquat?1,1'-ethylene-2,2'-dipyridylium,DQ?is a nonselective and defoliant contact herbicide that belongs to the bipyridinium class.The herbicidal effect is similar to paraquat?PQ?.In recent years,with the discontinuation of paraquat,the application of DQ in agriculture is increasing gradually.At the same time,the incidence of DQ poisoning is also simultaneously increasing.DQ poisoning will become a major challenge for clinicians after PQ poisoning.So far,the mechanism of DQ posioning has not been fully clarified.The aim of this study was to observe the pathological changes of lung,liver and kidney of rats at different dose and time point and determine the optimal dose of DQ poisoning by establishing the rat model of DQ poisoning in different degrees.Meanwhile,a simple and sensitive HPLC method were established and verified for quantifying diquat concentration in plasma and tissues.The toxicokinetics and tissue distribution of DQ in Wistar rats were studied by single oral?single dose?administration in order to provide experimental data for further study of acute poisoning of DQ.Part one Establishment and evaluation of acute diquat poisoning model in Wistar ratsObjective:To establish the Wistar rat model of acute diquat poisoning and observe the pathological damage of main target organs.Methods:Thirty-six Wistar rats were randomly divided into six groups?n=6?,including one normal saline control group.and five treatment groups which were separately given single-dose of intragastric administration at the doses of46.2 mg/kg,77 mg/kg,115.5 mg/kg,231 mg/kg and 346.5mg/kg.The pathological changes of lung,liver and kidney were observed by hematoxylin and eosin?HE?and Masson staining.The optimal dose was determined according to the general situation and pathological changes.Thirty-six Wistar rats were randomly divided into five treatment groups and one normal saline control group.Treatment groups were given single-dose of intragastric administration according to the optimal dose.The rats were sacrificed at 1st,3rd,7th,11th and 14th day after exposed,respectively.The activity of serum glutamic-pyruvic transaminase?ALT?and glutamic-oxalacetic transaminase?AST?were measured by chemical colorimetry.The pathological changes of lung,liver and kidney were observed by HE and Masson staining.Results:Compared with the control group,there was no obvious symptom in the low dose group?46.2mg/kg and 77mg/kg group?.The 14d survival rate was100%.115.5mg/kg group on the first day showed lassitude,reduced intake of food and water,the sloppy soft stool.On the third day of exposure,mild hyperemia appeared in the eyeball and mild blood secretion around the eye and nose was occasionally seen.The symptoms began to relieve on the 4th day of exposure,and the 14d survival rate was 100%.The high dose group?231mg/kg and 346.5mg/kg groups?on the first day showed lassitude,reduced intake of food and water,shallow and rapid breathing and diarrhoea.On the second day of exposure,hyperemia appeared in the eyeball,blood secretion around the eye and nose and slow breathing depth were seen.The symptoms began to relieve on the 7th day of exposure,and the 14d survival rate was66.7%and 50%,respectively.Taking 115.5mg/kg as the best dose,it was found that the serum AST and ALT activity of rats on the first and third day of exposure was significant higher than those in control group.The results of pathological examination exhibited that in low dose group?46.2mg/kg and77mg/kg group?,on the 14th day of exposure,the pathological changes of lung,liver and kidney were not obvious.In the high dose group?231mg/kg and 346.5mg/kg groups?,the normal structure of lung tissue disappeared,the proliferation of collagen fibers in the bronchi and alveolar septum was obvious,the renal tubules were focal necrosis,exfoliation,erythrocyte and clear tubular type were observed.The normal structure of hepatocytes disappeared,with vacuolar degeneration and punctate necrosis.In 115.5mg/kg group,the pathological changes of lung,liver and kidney began to appear on the first day of exposure,the pathological changes were the most serious on the third day,and then gradually alleviated.On the 14th day,the alveolar septum was slightly widened,with inflammatory cell infiltration,local alveolar cavity became narrow,atrophy,peripheral alveolar compensation,bronchi and alveolar septum collagen fiber proliferation;The local renal tubular epithelial cells were enlarged and necrotic;the central vein surrounding hepatic cells showed vacuolar degeneration with punctate necrosis.Conclusion:The rat model of acute diquat poisoning can be successfully induced by single-dose of intragastric administration.The condition of wistar rats and the pathological damage of the main target organs could be observed during the whole course of 115.5mg/kg administration.Part two Study on the Toxicokinetics and tissue distribution of diquatObjective:To establish and verify a simple and sensitive HPLC method for quantifying diquat concentration in plasma and tissues.Methods:Chromatographic separation was performed on a PC HILIC S5 column?150mm×2.0mm I.D.,5?m?.The mobile phase consisted of 20mmol ammonium formate aqueous solution containing 0.1%formic acid?A?and acetonitrile?B?.The flow rate was 0.3ml/min.Mass spectrometric analysis was conducted on a QTRAP 5500 triple quadrupole mass spectrometer?AB SCIEX,USA?equipped with an ESI source.Mass spectrometric analysis was performed on the positive ion MRM?multiple reactions monitoring?mode.The mass transitions of DQ?m/z 183.0>157.1?and EV?m/z 214.0>185.0?were optimized.In the study of toxicokinetics,twenty-four fasted male Wistar rats were randomized into four groups.Blood samples were collected from rats at 4 time points in each group.The animals were administered115.5mg/kg DQ?1ml/100g BW?by single-dose of intragastric administration.Blood samples were collected into heparinized Eppendorf tubes through collection from the retrobulbar venous plexus of rats with the aid of heparinized glass capillaries at 14 time points of pre-dose and after dose of5min,10min,25min,40min,1,2.5,5,8,10,11,12,13,16,24 and 34h.Plasma was obtained from blood samples by centrifuging and immediately frozen at-80?for toxicokinetic analysis.In the study of tissue distribution,eighteen fasted male Wistar rats were randomized into three groups.The animals were administered 115.5mg/kg DQ?1ml/100g BW?by single-dose of intragastric administration and were euthanized using chloral at 25min,1h and24h after dosing.The heart,liver,spleen,lung,kidneys,stomach,small intestines,brain and muscles tissues were separately collected and immediately frozen at-80?for tissue distribution studies.Results:The calibration curves of DQ obtained from extraction of plasma and tissues were liner over the range from 10¹000ng/ml and the LLOQ of DQ was 10ng/ml.The intra-day and inter-day precision?%RSD?was less than10.42%,and the accuracy was 96.42-111.63%.The recoveries of diquat in plasma and tissue were between 87.4¹10%and 94.1¹1.3%,respectively.The results prove that DQ in plasma and tissue could maintain the stability after exposure at different conditions.The toxicokinetics parameters were calculated using DAS 3.2.8 by a non-compartmental statistical matrix method.After oral administration,DQ was quickly adsorbed and the average time at maximum concentration(t_max)was 0.9±0.3h.The maximum concentration?Cmax?was 5287.5±2189.5ng/ml.The average half-life(t_1/2)and mean retention time?MRT?were 19.3±4.3h and 4.9±0.7h,respectively,which showed there existed a slower elimination phase.The area under the concentration-time curve(AUC_0-?)was 13024.5±3305.7mg/h/L.The results of tissue distribution showed that diquat could be rapidly distributed to various tissues,which were mainly distributed in the small intestine,stomach and kidney.In addition,the heart,lung,spleen,brain and skeletal muscle contained a small amount.But DQ was not detected in the liver.Conclusions:Diquat is quickly absorbed in vivo and reaches the peak at 1 hour after dosing.It is widely distributed and mainly distributed in the small intestine,stomach and kidney,followed by the heart,lung,spleen,brain and skeletal muscle.