| There are reports of mushrooms poisoning all over the world.In China,mushroom poisoning which has the highest mortality,has become the first cause of death in food poisoning incidents.Mushrooms containing amanita peptide toxins were responsible for more than 90%of deaths in mushroom poisoning.In recent years,scientists had made great progress in detection of amanita peptide toxin and identification of mushrooms.It has been found that the species of mushrooms containing amanita peptide toxin in China were quite different from those in Europe and America.From 2000 to 2014,Amanita exitialis,an unique deadly Amanita species in China,had been identified in more than 25 mushroom poisoning incidents,with 99 people poisoned and 48 deaths.The poisoning incident caused by Amanita exitialis had been first reported in Guangdong province.However,there have been few case report of A.exitialis poisonings with systematic data.Thus,the clinical feture of A.exitialis poisonings is lacking.There is no special antidote in the treatment of patients poisoned with mushrooms containing amanita peptide toxin.In hope for changing the toxicokinetics process of amanita peptide toxin in the body,gastric lavage,activated charcoal,diuresis,bile drainage and blood purification techniques are conventional clinical managements.Due to the absence of exposure markers in clinical practice and the lacking of appropriate animal models,the dynamic changes and excretion characteristics of the amanita peptide toxin are not completely clear.Thus,the above treatment methods lacked the corresponding theoretical basis on toxicokinetics study in amanita peptide toxin.Hence,it is necessary to carry out the following two parts of studies:(1)By investigating of mushroom poisoning events and collecting A.exitialis poisoning cases,we discussed the clinical characteristics of toxic liver damage caused by mushrooms containing amanita peptide toxin in China.(2)By establishing hepatotoxic model induced by Amanita exitialis in beagles and the long-term bile drainage model of beagle dog,we described the dynamic changes in blood and excretion process of the three amanita peptide toxins in beagle dogs,we calculated the kinetic parameters of the three amanita peptide toxins and found features of the three amanita peptide toxins,in hope for providing theoretical basis for the clinical toxin clearance treatment.Part I Study on the clinical characteristics of mushroom poisoning caused by A.exitialisObjective:By investigating of mushroom poisoning events and collecting A.exitialis poisoning cases,we discussed the clinical characteristics of toxic liver damage caused by mushrooms containing amanita peptide toxin in China.Methods:We investigated three A.exitialis mushroom poisoning cohorts in Yunnan Province in 2014 and 2015,involving 10 patients.5 mushroom samples,25 blood samples and 5 urine samples were collected.Mushroom samples were identified by morphological and molecular studies.UPLC-ESI-MS-MS method was used to detect the peptide toxins in the mushroom,blood and urine samples.Epidemiological information,clinical data,and results of laboratory examinations were collected and analyzed.Results:The mushroom samples were all identified as A,exitialis in sect.Amanita.The average toxin concentration decreased from the cap to the stipe to the volva,and the average concentration of the peptide toxins decreased in the order of a-amanitin>Phallacidin>β-amanitin>y-amanitin.The latency period between ingestion and the onset of symptoms was 13.9±2.1 h,and the time from ingestion to hospitalization was 49.6±8.5 h.The most common symptoms were nausea and vomiting(100%).Four patients died from fulminant hepatic failure.On day 2 post-ingestion,the level of ALT,AST,TBIL,DBIL,PT and APTT increased significantly(ALT 653.3 U/L;AST 509.8 U/L;TBIL 43.9 μgmol/L;DBIL 23.1 μmol/L;PT 23.7 s;APTT 43.8 s).On day 3 post-ingestion,ALT,AST,PT and APTT reached their peak levels(ALT 1018.6 U/L;AST 903.4 U/L;PT 49.8 s;APTT 79.8 s).On day 7 post-ingestion,the TBIL and DBIL levels reached the peak(TBIL 88.8 mol/L;DBIL 83 mol/L).After reaching the peak,the levels of ALT,AST,TBIL,DBIL,PT and APTT gradually decreased,and returned to their normal levels on day 14 after ingestion.values were at normal levels.The death group and the survival group had a similar variation trend of serological indexes,but the death group had a greater change.After 48 h ingestion of A.exitialis,amanita peptide toxins can not be detected in the blood of the patients.Within 96 h ingestion of A.exitialis,amanita peptide toxins can be detected in the urine in some of the patients.Conclusion:A.exitialis is nowrecognized as an extremely dangerous mushroom.This study reveals that poisoning by A.exitialis is characterized by the long latency before the onset of(6-24 h post-ingestion)GI symptoms and subsequent liver damage characteristic of amatoxin poisoning.Detection of amanita peptide toxins in blood within 48 h and in urine within 96 h showed diagnostic value in Amanita peptide toxins poisoning.Part Ⅱ Preliminary study on toxicokinetics of Amanita peptide toxins in beagles2.1 Establishing hepatotoxic model induced by Amanita exitialis and the long-term bile drainage model in beagle dogsObjective:Establishing hepatotoxic model induced by Amanita exitialis in beagles,we hope for providing help to studies on diagnosis and treatment of poisoning with amanita peptide toxins.Establishing long-term bile drainage model of beagle dogs,we hope for providing method to studies on bile secretion in bile-related diseases including amatoxin poisoning.Methods:12 male beagle dogs were randomly divided into 60 mg/kg group and biliary drainage group of six animals each.60 mg/kg group was used to establish hepatotoxic model induced by Amanita exitialis in beagles.Bile drainage group was used to long-term bile drainage model of beagle dogs.In 60 mg/kg group,UPLC-ESI-MS-MS method was used to detect peptide toxins in Amanita exitialis.To establish hepatotoxic model,the beagles were fed with 60 mg/kg of lyophilized powder of A.exitialis fungus which encapsulated in starch capsules.Toxic sighs were observed,coagulation function,hepatic and renal function,liver histopathological morphology,peptide toxin concentration in plasma and urine were detected during the experiment.In biliary drainage group,after accepting biliary drainage operation,the dogs were dressed in homemade underwear,coats,and Elizabeth circles for a week to ensure pre-experimental acclimatization.Surgical incision and general condition of beagle dogs were observed everyday.Bile and urine volumes of each day were collected and recorded.The changes of coagulation and liver function in beagles after operation were measured.The beagles in bile drainage group were executed at day 28 after operation,and liver tissues were taken for pathological examination.Results:Total peptide toxins in Amanita exitialis was 3482.6±124.94 mg/kg.In 60 mg/kg group,the beagles had toxic signs including vomiting and diarrhea in 12-48 h after ingestion.On 24 h after ingestion,the beagles’ ALT,AST,TBIL model,ALP,PT and APTT levels increased obviously.On 36 h after ingestion,the beagles’ ALT,AST,PT and APTT values reached their peaks(ALT:283.2 Kallmann unit;AST:125.6 Kallmann units;PT:132.9 s;APTT:131.4 s).On 48 h after ingestion,the beagles’TBIL and ALP levels reached their peaks(TBIL:23.3 mol/L;ALP:274.5 U/L).The beagles’TBIL,TP and APTT returned to normal 1 week after ingestion,their ALT,AST and ALP levels returned to normal 3 weeks after ingestion.Three dogs died during 24-72 h after ingestion.Liver histopathological morphology study showed hemorrhagic necrosis of hepatocytes.Peptide toxins can be detected in plasma with 24 h after ingestion.Peptide toxins can be detected in urine with 92h after ingestion.In the bile drainage group,no wound dehiscence was observed after stitch removal at day 7 after the operation of the beagles.Weakness,loss of appetite,physical inactivity were observed in the dogs within 2 days after operation.The dogs became nomal on the fourth day after operation.The level of ALT,AST and ALP on 1 d,2 d and 3 d after operation in beagle dogs were statistically higher than those before operation(P<0.05),and these level became to normal a week after operation.There was no statistically differences in TBIL,DBIL,PT,APTT,BUN and CRE values at different time points in biliary drainage of beagles,compared with those before operation(P>0.05).There were no obvious pathological changes in the livers of the Beagle dog.The daily urine volume of the bile drainage group was 250.4± 19.1 mL/d,and the daily bile flow rate was 69.9±6.1 mL.Conclusion:Amanita peptide toxins can cause hemorrhagic necrosis of liver cells and lead to acute liver failure.This model is consistent with clinical pathophysiological process of toxic liver damage induced by mushrooms containing peptide toxins and the detection time limit of peptide toxins in urine and blood.And it can be applied to the study of diagnosis and treatment of poisoning induced by mushrooms containing peptide toxins.The long-term bile drainage model can be used to monitor the bile excretion of the dog with beagles in a normal state(at least 28 d).This model can be applied to study bile secretion and components in bile-related diseases including amatoxin poisoning.2.2 Study on dynamic changes in blood and excretion of Amanita peptide toxins in beaglesObjective:By describing the dynamic changes in blood and excretion process of the three amanita peptide toxins in beagle dogs,we calculated the kinetic parameters of the three amanita peptide toxins and found features of the three amanita peptide toxins,in hope for providing theoretical basis for the clinical toxin clearance treatment.Methods:36 male beagles were randomly divided into control group,bile drainage group,20 mg/kg group,20 mg/kg bile drainage group,60 mg/kg group and 60 mg/kg bile drainage group,with 6 dogs in each group.In 20 mg/kg group and 60 mg/kg group,the beagle dogs were fed with 20 mg/kg and 60 mg/kg of lyophilized powder of A.exitialis fungus which encapsulated in starch capsules.In 20 mg/kg bile drainage group and 60 mg/kg bile drainage group,the beagle dogs were fed with 20 mg/kg and 60 mg/kg of lyophilized powder of A.exitialis on the eighth day after bile drainage operation.In control group and bile drainage group,the beagle dogs were fed with empty capsules.They were assessed for toxicity signs and biochemical and pathological changes.UPLC-ESI-MS-MS method was used to assay peptide toxins in mushroom,blood,urine,bile and feces samples at different time points.By comparing the differences in toxicokinetic parameters and excretion of peptide toxins between the dogs with bile drainage and the dogs without bile drainage,we analyzed the role of bile drainage in the process of poisoning induced by mushrooms containing amanita peptide toxins.Results:No toxic sighs and death was observed in control group,biliary drainage group and the 20 mg/kg bile drainage group.After 12-48 h,33.3%of the dogs in 20 mg/kg group,100%of the dogs in 60 mg/kg group and 50%of the dogs in 60 mg/kg bile drainage group had vomiting and diarrhea.Three dogs died in 60 mg/kg group,and no death was observed in other experimental groups.In 20 mg/kg group and 60 mg/kg group,on 24 h after ingestion,the beagles’ ALT,AST,TBIL model,ALP,PT and APTT levels increased obviously.On 36 h after ingestion,the beagles’ ALT,AST,PT and APTT values reached their peaks.On 48 h after ingestion,the beagles’ TBIL and ALP levels reached their peaks.The beagles’ TBIL,TP and APTT returned to normal 1 week after ingestion,their ALT,AST and ALP levels returned to normal 3 weeks after ingestion.During the experimental period,the levels of BUN and CRE were normal in all experimental groups.After A.exitialis ingestion at the same dose,the beagles with biliary drainage showed less severe toxicity signs and slighter biochemical and changes than the beagles without biliary drainage.In 60 mg/kg group,liver histopathological morphology study showed hemorrhagic necrosis of hepatocytes,and there were no obvious pathological changes in the livers of the beagle dog in the other experimental groups.The main toxicokinetic parameters of the three peptide toxins were as follows:Tmax was between 1.25 and 2.08 h,MRT was between 1.95 and 3.82 h,T1/2 was between 0.54 and 1.94 h,CLs was between 0.12 and 0.54 L/h/kg,Vz/F of a-Amanitin and β-Amanitin was between 0.04 and 0.12 L/kg,Vz/F of Phallacidin was 0.43 L/kg.Three peptide toxins in the 0-2 d urine accounted for more than 90%of the total urine excretion.Three peptide toxins in the 0-1 d feces accounted for more than 90%of the total feces excretion.Three peptide toxins in bile accounted for less than 20%of the total excretion in the urine and bile.After A.exitialis ingestion at the same dose,the beagles with biliary drainage showed less severe toxicity signs and biochemical and pathological changes,much lower internal exposure and accumulated amounts of peptide toxins in the urine,much higher amounts of peptide toxins in the feces than beagles without biliary drainage.Conclusion:a-Amanitin,β-Amanitin and phallacidin were rapidly absorbed through the gastrointestinal tract and eliminated from plasma after A.exitialis was ingested.Gastric lavage and enhanced plasma clearance methods such as hemodialysis,hemoperfusion,or plasma exchange may be ineffective in removing amatoxins from blood before 12 h post ingestion.a-Amanitin and P-Amanitin are mainly distributed in extracellular fluid,and Phallacidin is more widely distributed in the body.Although amatoxins in bile accounted for less than 20%of the total excretion in the urine and bile,but the toxin in bile comes from hepatocytes.This portion of toxin is the active toxins causing hepatotoxicity and may aggravate hepatocyte damage,and it is still unable to judge whether bile drainage is effective.Bile promotes the intestinal absorption of amanita peptide toxins,but the mechanism is unclear. |
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