The Protective Effects And Possible Mechanisms Of Diallyl Trisulfide(DATS) On Leukopenia Induced By Benzene

ObjectiveBenzene is a well-known occupational and environmental toxicant,and was identified as an environmental carcinogen by the International Agency for Research on Cancer(IARC).Benzene has been regarded as the most important reason of chronic occupational poisoning.Benzene exposure to the general population originates from environmental contamination of ambient air,such as cigarette smoke,gasoline vapours,and automobile emissions.Therefore,benzene exposure has become an increasing environmental concern for the health of workers and the general public.Haematological diseases,such as cytopenia,aplastic anaemia,leukopenia and leukaemia are associated with benzene exposure,and the early symptom is characterized by leukopenia.However,there is no evidence of a threshold.An epidemiological study claimed that the white blood cell and platelet counts in peripheral blood of workers exposed to benzene(even below 1 ppm in air)were significantly lower than those observed in a control group of people.However,no effective nutritional preventive strategy has been developed to date.Therefore,benzene exposure has become an increasing environmental concern for the health of workers and the general public.When garlic is chopped or crushed,alliin is released and produces allicin catalyzed by allinase enzyme,which further converts several organosulfur compounds(OSC),including allicin(ALI),diallyl sulfide(DAS),diallyl disulfide(DADS),diallyl trisulfide(DATS),allylmethylsulfide(SAC)and S-allyl mercapto cysteine(SAMC).It is known that organic sulfides in garlic have biological activity such as anti-oxidation,anti-inflammatory,anti-tumor,anti-atherosclerosis,antibacterial and chemical protection.In particular,garlic organic sulfides can play a role in chemical protective effect by promoting the detoxification and discharge of chemical poisons.Wang Shuo et al.found that diallyl trisulfide can suppress n-hexane induced peripheral nerve injury through regulating metabolic enzyme cytochrome P450.Zeng Tao’s study showed that garlic oil(garlic liposoluble extract)inhibited leukopenia induced by chemotherapy and radiotherapy in tumor-bearing mice.Our previous study also found that garlic oil can inhibit benzene-induced leukopenia,the effective compound in garlic oil responsible for the protective effect against leukopenia and its underlying mechanisms remain unclear.And also it has been suggested that the number of sulfur atoms is critical in the biological activities of organosulfur compounds in garlic.Therefore,in this study,DATS which contains three organic sulfur groups in garlic extract,was used as an intervention drug and oral administration of benzene was used to establish the benzene poisoning model.In order to observe the effects of DATS against leukopenia and reveal the possible mechanism,we designed the study in four aspects including anti-cell apoptosis,anti-oxidation and regulation of benzene metabolism and PI3K/Nrf2/ARE pathway.The results were expected to provide theoretical basis for the development of pharmacological effects of DATS,and provide nutrition control strategies for the prevention and treatment of chronic benzene poisoning.Methods1.To observe the preventive effect of three organic sulfur preparations of garlic on benzene-induced leukopenia.Kunming mice were divided into 11 groups:blank control group,model group,garlic homogenate(GH)low and high dose intervention group,GH control group,garlic oil(GO)low and high dose intervention group,GO control group,diallyl trisulfide low,high dose intervention group and DATS control group.Except the blank control group and the model group,the others were administrated by gavage with GH,GO or DATS.The counts of white blood cells(WBC)in peripheral blood were detected to identify the preventive effect of three preparation,and organ index and pathological examinations of liver and spleen were used to observe the protective effects and potent adverse events of three preparation.2.Effects of PI3K-Akt pathway-mediated apoptosis and antioxidation of DATS against benzene-induced leukopenia.60 male SD rats were randomly divided into 6 groups:blank control group,benzene poisoning model group,DATS low,middle and high intervention group and DATS control group,10 rats in each group.DATS(15,30,45mg/kg BW)was given to DATS groups respectively.After 2 hours,except the two control groups treated with the same volume of corn oil,the rats in the other four groups were given benzene-corn oil suspension(1.3 g/kg BW)to build the benzene poisoning model,once a day.28 days later,blood was collected from abdominal aorta,peripheral blood cell counts were detected.The peripheral blood mononuclear cells and bone marrow cells were collected for evaluating cell apoptosis.The expression of apoptosis-related protein and PI3K-Akt pathway related protein were detected by Western blotting.Reactive oxygen(ROS)and malondialdehyde(MDA)were measured to determine the level of oxidative stress.The levels of non-enzymatic antioxidant reduced glutathione(GSH),antioxidant enzyme superoxide dismutase(SOD),Glutathione peroxidase(GPx)and glutathione reductase(GR)activity were used to assess the antioxidant capacity.3.The role of regulation of benzene metabolism in the effect of DATS against benzene-induced leukopenia50 male SD rats were randomly divided into 5 groups:blank control group,benzene poisoning model group,DATS low and high intervention group and DATS control group,DATS intervention groups and control group were treated with different doses of DATS(15,30mg/kg.BW),the other groups were given the same volume of corn oil.After 2h,except the control groups,the others were given benzene(1.3 g/kg)by oral administration establish the benzene-induced leukopenia model,once a day for 28 days.S-phenylmercapturic acid(SPMA)and trans,trans-muconic acid(t,t-MA)were measured by high performance liquid chromatography(HPLC).The blood parameters were examined to identify specific changes of benzene-induced toxicity.Western blot and chemical method were used to detect the activities and protein expression levels of enzymes CYP2E1 and glutathione-S-transferase(GST)in liver and enzymes myeloperoxidase(MPO)and NADPH:quinone(NQO1)in bone marrow.4.The role of PI3K/Nrf2/ARE signaling pathway-mediated phase-Ⅱ metabolic enzymes and antioxidant enzymes in DATS suppressing benzene-induced leukopenia.60 male SD rats were randomly divided into four groups:blank control group,benzene poisoning model group,DATS intervention group and DATS control group,15 rats in each group.DATS(30 mg/kg BW)was administered orally to the DATS intervention group and the DATS control group.After 2 h,the rats in the model group and the DATS intervention group were treated with oral benzene-corn oil suspension(1.3 g/kg BW),and others were given corn oil with same volume,once a day for 28 days.The levels of ROS and MDA were measured to assess the level of oxidative stress.The expression of NQO1,GST,SOD and GPx were determined by Western blot.The expression levels of PI3K/Nrf2/ARE signaling pathway-related proteins:p-PI3K,p-Akt,Nrf2 and Keapl were detected to evaluate the regulation effect of PI3K/Nrf2/ARE signal pathway and downstream were analyzed and downstream genes.Results1.To observe the preventive effect of three organic sulfur preparations of garlic on benzene-induced leukopenia.The results showed that the WBC of mice in the model group was significantly decreased to 41.31%(p<0.01)compared with that in the blank control group.After intervention by GH(400 mg/kg),GO(60 mg/kg)and DATS(30 mg/kg),the WBC increased by 144.04%,140.07%and 148.34%,respectively.But there was no statistically difference of WBC between the GH,GO and DATS control groups and the blank control group.The spleen index and thymus index of mice in the benzene model group were 0.55 and 0.45 lower than those in the blank control group,and the number of spleen nodules was reduced and the thymus was atrophy.However,there was no statistically difference of the liver index and pathological examination among all groups.In GH(400mg/kg)and GO(60mg/kg)control group,the spleen and the spleen index of the mice were 1.32 and 1.29 times higher than those in the blank control group,respectively.2.Effects of PI3K-Akt pathway-mediated apoptosis and antioxidation of DATS against benzene-induced leukopenia.Our results demonstrated that the counts of peripheral blood WBC and RBC decreased to 31.0%and 79.2%,respectively,in the benzene poisoning model group compared to the control.However,blood cell counts were restored by DATS treatment(30 mg/kg,45 mg/kg).The apoptosis rates of peripheral blood mononuclear cells(PBMCs)and bone marrow cells(BMCs)were increased to 274%and 284%,respectively,following benzene exposure.Furthermore,expression levels of Bcl-2,p-PI3K and p-Akt were downregulated and those of Bax were upregulated in both cell types.Interestingly,DATS treatment can restore the WBC number by 267.1%and 304.8%while RBC number by 108.6%and 117.7%in 30,45 mg/k DATS treated groups.Moreover,the levels of ROS and MDA of PBMCs were 3.4 and 1.5 times higher and the levels of ROS and MDA in BMCs were 3.3 and 2.2 times higher in the model group than those in the control group,respectively,while the level of non-enzymatic antioxidant GSH in PBMCs and BMCs decreased by 42.7%and 45.2%,respectively,and the activities of antioxidase(SOD,GPx and GR)were also decreased.After DATS intervention,the oxidative stress parameters in the cells decreased,the activity of antioxidase increased,and the GSH content increased with a dose-dependent manner.3.The role of regulation of benzene metabolism in the effect of DATS against benzene-induced leukopeniaThe WBC was reduced by 65.5%after benzene exposure compared to that in the blank control group,and two benzene metabolites could be detected in the urine.The levels of t,t-MA in urine decreased by 19.84%and 53.68%,respectively,and the level of SPMA increased by 0.55 times and 0.92 times,respectively.The results showed that DATS regulated benzene metabolism.Meanwhile,the results of activities of enzymes reported that activities of CYP2E1 and MPO were 1.80 and 3.03 times were higher than those in blank control group,while activities of GST-1 and NQO1 decreased to 70.53%and 42.44%after benzene exposure(p<0.001),which were reversed by DATS which were supported by the results of Western blot tests of protein expression of these enzymes.The activities and protein expression of GST-1 and NQO1 in DATS control group were significantly higher than those in control group,while those of CYP2E1 were significant lower(p<0.05).4.The role of PI3K/Nrf2/ARE signaling pathway-mediated phase-Ⅱ metabolic enzymes and antioxidant enzymes in DATS suppressing benzene-induced leukopenia.In the model group,the WBC of benzene model group decreased by 67.66%and RBC decreased by 20.8%compared with blank control group.After DATS intervention,WBC and RBC were 1.56 times and 0.11 times higher than those of model group.The levels of oxidative stress of PBMCs and BMCs in benzene model group increased by 2.45 and 2.08 times,and the levels of MDA increased by 2.23 and 1.48 times,respectively.The protein expression levels of antioxidant enzymes(SOD and GPx)and phase-ⅡI(GST and NQO1)decreased.After DATS treatment,oxidative stress levels decreased,the protein expression of antioxidant enzymes and phase Ⅱ metabolic enzyme were down-regulated.The expression of p-PI3K,p-Akt and Nrf2 protein was up-regulated,the expression of Keapl protein was down-regulated and PI3K/Nrf2/ARE signal pathway was activated.Conclusions1.Garlic homogenate,garlic oil and DATS all can effectively suppress benzene-induced leukopenia,among which DATS is the safest and most effective nutrition preventive strategy.2.DATS treatment could suppress the benzene-induced oxidative stress by activating antioxidative enzymes and enhancing non-enzymatic antioxidant in PBMCs and BMCs3.Benzene-induced leukopenia may be related to the apoptosis of PBMCs and BMCs mediated by oxidative stress and DATS can prevent benzene-induced leukopenia by suppressing oxidative stress-mediated cell apoptosis via the PI3K/Akt pathway.4.The inhibition of bioactivation in liver and bone marrow catalyzed by CYP2E1 and MPO and the activation of detoxification catalyzed by GST and NQO1 may be the critical mechanism by which DATS regulates benzene metabolism against the leucopenia induced by benzene.5.DATS regulates antioxidase-mediated antioxidant activity and promotes the phase-Ⅱ metabolism enzymes-mediated detoxification through regulating PI3/Nrf2/ARE signaling pathway,and thereby suppresses benzene-induced blood toxicity and cytotoxicity.