Evaluation Of The Subchronic Toxicity Of Diallyl Trisulfide And Its Effect On The Toxicity Of Cyclophosphamide

ObjectiveWorld Health Organization(WHO)statistics showed that cancer is the world’s chronic non-communicable diseases,second only to cardiovascular disease,which result in death in human being.In the past decade in China,the incidence of cancer is on the rise,seriously endangering the physical and mental health of the human body.At present,chemotherapy is the main means of cancer treatment.Cyclophosphamide(CTX),as a clinical chemotherapeutic agent,has a significant effect on the treatment of cancer,but it often kills normal cells at the same time while killing cancer cells,resulting in various toxic side effects,such as reducing white blood cell levels,immune inhibition,myelosuppression,gastrointestinal symptoms and apathetic,fatigue,hair loss,allergies and other manifestations.In China,to reduce the toxicity caused by chemotherapy,the clinical use of chemotherapy drugs in the treatment of tumors are often combined with traditional Chinese medicine extract or traditional Chinese medicine preparations at the same time,achieving some potential protective effect.However,there are still many problems with the use of traditional Chinese medicine,and its numerous drug types limit the selection of effective drugs.Garlic(Allium sativum L.)is amongst the oldest plants cultivated for its utilization as food and medicine.It has a variety of bioactive and nutrient ingredients,widely used in prophylactic as well as therapeutic purposes and health care.Various studies have showed that it is an incredible plant with numerous useful impacts such as anti-bacterial,anti-inflammatory,hypoglycemic and hypolipidemic activities,lowering blood pressure,prevetion of platelet aggregation,prevetion of cardiovascular disease and anti-tumor and so on.At the same time,the study revealed that the medicinal values of garlic are associated with a variety of sulfur compounds which are mainly contained or metabolized from garlic.Diallyl Trisulfide(DATS),the main sulfur-containing compound extracted from garlic,is the main active ingredient of garlic.Epidemiological investigation and experimental study indicate that DATS showed a preventive effect on gastric,colon,breast,prostate and esophageal cancer and other cancers in the human body,animal,cell levels.The mechanism of action mainly includes inhibition of tumor cells,induction of tumor cell apoptosis;inhibition of tumor cell metastasis and infiltration;cell cycle block regulation;antioxidant,etc.However,its effect on the toxicity of cyclophosphamide and subchronic toxicity are rare.Therefore,first we observed the subchronic toxicity of DATS on Wistar rats,which are oral administrated for 90 days.Further we established a hepatocellular carcinoma bearing mice model of H22,adopting CTX as a chemotherapeutic drug model,and DATS was selected for antitumor intervention,to determine the anti-tumor effect of DATS and its effect on the toxicity caused by cyclophosphamide.Finally,we provided the experimental basis for the safety evaluation of DATS on subchronic toxicity,and provided the basis for the development of DATS,using as health food and adjuvant anti-tumor drugs.MethodsThe studies were carried out in two batches of animals,and the acclimation period of animals was 5 days prior to experiment.The first batch of 80 Wistar rats were randomly divided into 4 groups of 20 each,male and female half,i.e.control,30,60 and 90mg/kg bw DATS(diluted in corn oil)respectively.DATS were given by oral administration with a volume of 0.5ml/100g.bw.Animals in control group were treated equal volume solvent per day for 90 days.During the experiment,the state and abnormal performance of the animals were observed and recorded daily,including general performance,behavior,poisoning and death.During the period,rats body weight was recorded once a week,and adding food twice a week,strictly record the amount of animal food intake and food residue,and calculate the food utilization rate.After oral administration for 90 days,red blood cell(RBC),hemoglobin(HGB),platelet(PLT),white blood cell(WBC)and its classification(lymphocytes,neutral cells,mononuclear cells)were measured after fasting for 16 hours.After oral administration of DATS for 90 days,5ml blood were collected in the centrifuge tube,placing at room temperature for 1 hour,3500r/min for 10min,collecting serum to determinate the aspartate aminotransferase activity(ALT),urea nitrogen(BUN),creatinine(CREA),total protein content(TP),albumin content(ALB),globulin content(GLB),total cholesterol CHOL),triglyceride content(TG)and glucose content(GLU)and other indicators.After the rats were sacrificed,the liver,kidney,spleen,stomach,ovaries or testes were weighed and the organ coefficients were calculated.5 animals were randomly selected from each group,and the liver,spleen,kidney,stomach,ovary or testicular parts were fixed in paraformaldehyde.HE staining was performed to detect pathological changes of animal organs in different DATS dose groups.The second batch of 75 male Kun-Ming tumor-bearing mice were established.H22 hepatocarcinoma cells were selected and washed with nomal saline(NS),1000r/min for 5min for 2 times.Then H22 cells were mixed with NS and 0.2ml suspension containing 2×107 cells/ml were subcutaneously injected at right axilla of each mice.After 24h,50 mice were randomly divided into five groups with 10 in each,i.e.model group,25mg/kg.bw CTX,50mg/kg.bw DATS,25、50mg/kg.bw DATS+CTX.After 24h,CTX dissolved in NS was intraperitoneally injected to mice in 25mg/kg.bw CTX group and 25、50mg/kg.bw DATS+CTX group once a day for consecutive 14 days,at a volume of 0.2ml/10g.bw.DATS diluted in corn oil were given by oral administration at the dosage of 25 mg/kg.bw and 50 mg/kg.bw DATS intervention groups respectively.Mice in medel group and CTX group were treated with equal volume solvent per day for 14 days.At the end of experiment,mice were scarificed by cervical dislocation.Blood was sampled into Eppendorf tube with EDTA2K to measure RBC,WBC and PLT.Then tumor,liver,spleen and thymus were weighted,calculating tumor inhibition rate the index of liver,spleen and thymus.ResultsIn experiment of first batch,compared with control group,the body weight of both male and female rats in 90mg/kg.bw DATS group was lowered from 8th week,the body weight and food intake in male 90mg/kg.bw DATS group decreased(p<0.05).Compared with control group,DATS significantly increased the level of WBC in male 30、60、90 mg/kg.bw DATS group(p<0.05),the level of WBC was increased in female 30 and 90 mg/kg.bw DATS group(p<0.05),and the level of PLT increased in male 90 mg/kg.bw DATS group(p<0.05).Compared with control group,the level of BUN increased in male 90 mg/kg.bw DATS group(p<0.01),the level of(GLU)significantly decreased in female 60、90 mg/kg.bw DATS group(p<0.05,p<0.01),and the level of TP,GLB and CHOL significantly increased in female 90 mg/kg.bw DATS group(p<0.05,p<0.01).Compared with control group,the weight of the liver was increased in male 60mg/kg.bw DATS dose group(p<0.05),the weight of spleen was increased in male 60、90mg/kg.bw DATS dose group(p<0.05,p<0.01).Compared with the control group,the liver index of the male and female 90mg/kg.bw DATS dose group was significantly increased(p<0.05,p<0.01).The spleen index in the male 60、90mg/kg.bw DATS group increased(p<0.05).The kidney index increased in female 30、60、90mg/kg.bw DATS group(p<0.01,p<0.05).HE staining demonstrated that DATS showed little toxicity to liver,stocmatch and ovarian/testisto.However,DATS showed toxicity to spleen and kidney to some degree.In experiment of second batch,no significant abnormality in body weight was detected in model group.Howerer,compared with model group,the body weight growth of 25mg/kg CTX were lower,at the end of experiment,the body weight in this group was markly decreaed(p<0.05).The body weight of 25mg/kg DATS+CTX was slightly higher than that of CTX group.Compared with model group,the tumor weight in 25mg/kg CTX reduced 69.8%(p<0.01),meanwhile,25、50mg/kg DATS+CTX caused 67.9%(p<0.01)and 69.8%(p<0.01).There is no significant difference among 25mg/kg.bw CTX,25mg/kg DATS+CTX and 50mg/kg DATS+CTX(p>0.05).Compared with the model group,the tumor inhibition rate was 32.0%(p>0.05)in the 50mg/kg DATS group,but there was significant difference compared with the CTX group(p<0.05).Compared with the model group,the WBC level of 25mg/kg.bw CTX group was significantly decreased(p<0.05),the level of WBC in both 25mg/kg DATS + CTX group and 50mg/kg DATS + CTX group slightly increased,however,there no significant difference compared with the CTX group(p>0.05).Compared with the model group,CTX chemotherapy had little effect on the weight of liver and kidney(p>0.05),but CTX chemotherapy could significantly decrease the spleen,thymus weight and organ index(p<0.05).Compared with 25mg/kg CTX group,25mg/kg DATS + CTX group and 50mg/kg DATS + CTX group could significantly increase the spleen and thymus weight,but no mark increase in spleen and thymus index(p>0.05).Compared with the model group,25mg/kg.bw CTX chemotherapy can significantly reduce the bone marrow DNA content(p<0.05),DATS intervention could increase the bone marrow DNA content to some degree,but compared with CTX group,the elevated level was not significant(p>0.05).Conclusions1.DATS increase the level of white blood cells(WBC)and spleen weight in the experiment of subchronic toxicity evaluation.2.DATS show anti-tumor effect to some degree;Combain application of DATS and CTX showed no synergistic effect in tumor inhibition.3.DATS could antagonize spleen weight loss on tumor-bearing mice induced by chemotherapy,but there no obvious antagonistic effect on white blood cells,thymus weight reduction and bone marrow suppression.