The Synthesis And Removing Aluminum Effect And The Mechanism Of DFP Protecting Against Chronic Aluminum-Exposed Animals

Objective;Aluminum(Al)is ubiquitous in the environment,and is extensively used in contemporary life.Food additive,drugs and utensils comprising of aluminum are main sources of aluminum uptake in China.Aluminum is a known chronic neurotoxicant. There is evidence that aluminum has been linked to neurodegenerative disorders including Alzheimer's Diseases(AD),dialysis encephalopathy(DE).In present,it can be affirmed that aluminium is the only factor associated with DE.Employees in aluminum refinery could obviously be caused mental dysfunction.Chelators have been useful to treat aluminium-overload diseases in clinical practice.So far,the only diagnosis of Al accumulation and treatment of Al toxicity available has been achieved with cbelater desferrioxamine(DFO).It was found that DFO can reduce Al-induced mortality associated with the dialysis encephalopathy syndrome,the trabecular bone Al,and can improve bone histomorphometry and Al-related bone disease.Because of the poor gastrointestinal absorption,it can be administered by slow subcutaneous perfusion,resulting in a reduced compliance of the therapy.The use of DFO in dialysis patients induces major side effects.In some patients,ocular toxicity, disturbances of colour vision and loss of hearing have been described.So a cheap, orally active and non-toxic aluminum chelator is needed. 1,2-dimethyl-3-hydroxypyrid -4-one(deferiprone,DFP)originally treatment of iron overload.Aluminum and iron are hard acids with similar ionic radii(54 and 64 pro). They bind to the same plasma proteins.In the following experiments,DFP is used as a new type of aluminum chelating agent.Fri st,the s ynthesis of DFP has been accomplished.Scond,toxicological experiment has been done by LD₅₀experiment, Peripheral blood and bone marrow cell micronucleus experiment,and Sperm aberration experiment.Third,The effect of DFP on aluminum mobilization and elimination from tissues and serum as well as the influence on the excretion of other trace elements in mice and rabbits was investigated.Fourth,the treatment mechanism of DFP in curing animals poisoned by aluminum is studied.Those studies can provide theoretical foundation for seeking a cheap,orally active and non-toxic aluminum chelator for aluminum overload.Methods1.The synthesis of DFPDFP is synthesized from 2-methyl-3-hydroxy-4-pyrones and Methylamine(CH₃NH₂) via heating at 95～100℃6.5h.2.Toxicological experiment2.1 LD₅₀experiment 50 healthy mice of Kunming strain(25 males and 25 females) were randomly divided into 5 groups according to their body weight.The animals were given the medicine solution with the lavage method and observed for one week, then the number of dead mice were recorded.2.2 Peripheral blood and bone marrow cell micronucleus experiment 120 healthy mice of Kunming strain(60 males and 60 females)were randomly divided into 5 groups according to their body weight;negative control group,Cyclophosphamide positive control group,1/8LD₅₀group,1/4LD₅₀group and 1/2LD₅₀group.Then DFP bone marrow cell micronucleus experiment 24 hours after the second and the seventh application of medicine were conducted.2.3 Sperm aberration experiment 150 healthy male mice of Kunming strain were randomly divided into 5 groups according to their weight;negative control group, Cyclophosphamide positive control group,1/8LD₅₀group,1/4LD₅₀group,1/2LD₅₀ group.The medicine was applied for 5 successive days.On the 14th,28th,35th,42nd and 70th day after the application of medicine,DFP Sperm aberration experiment was carried out.3.Effect of DFP removing aluminum3.1 Effect of DFP removing aluminum in mice 84 healthy Kunming mice,which were different in sex,were randomly divided into 6 groups by body weight;Negative control group,Al-exposed group,preventive group low-dose group,medium-dose group,and high-dose group.The mice were given food and water ad libitum.The mice were sacrificed by decapitation at the end of the treatment.The serum samples were obtained by collecting and centrafugating blood samples.The selected organs(liver,kidney,brain and bone)were removed,weighted,and frozen for later analysis of aluminum,copper,zinc,calcium,iron and magnesium contents by atomic absorption speetrophotometry.3.2 Effect of DFP removing aluminum in rabbits3.2.1 Acute removing aluminumTwenty four New Zealand white rabbits were treated with aluminum.Rabbits were randomly divided into four groups;Al-only,low-dose group,medium-dose group, and high-dose group.DFP(500μmol/kg),DFP(750μmol/kg),DFP(1000μmol/kg)were given intragastrically(ig)respectively.Urine was collected hourly for 6h and analyzed for aluminum and essential elements copper,zinc and manganese.3.2.2 Combination with Vc or Mesna on urinary aluminum excretion Twenty four New Zealand white rabbits were treated with aluminum.Rabbits were randomly assigned to four groups;Al-only,DFP,DFP +Vc,DFP +Mesna.The agents mentioned above were given intragastrically.Urine was collected for six hours and the aluminum concentration was determined by atomic absorption spectrophotometry.3.2.3 Comparison of the efficacy of removing aluminum with various administration of DFP Eighteen New Zealand white rabbits were treated with aluminum.Rabbits were randomly assigned to three groups;Al-only,injection group, intragastrically group.DFP was given by subcutaneous injection and intragastrically respectively.Urine was collected for six hours and the aluminum concentration was determined by atomic absorption spectrophotometry.3.2.4 Subchronic removing aluminum Sixteen New Zealand rabbits were randomly divided into three groups;control,Al-only and DFP+Al.The Al-only and DFP+Al animals received injections of aluminum for three weeks.One week after the last Al injection the DFP+Al rabbits were given DFP intragastfically for 2 weeks.At the 42th day the animals were sacrificed and the organs were taken and digested. Blood was taken from the ear artery 3 times(at the initiation of the experiment, before and after DFP administration).The aluminum and copper,zinc,manganese were determined by atomic absorption spectrophotometry.4.Study on the mechanism of DFP protecting against chronic aluminum-exposed animals4.1 Effect of recovering the anti-oxidizing system 84 healthy Kunming mice, which were different in sex,were randomly divided into 6 groups by body weight; Negative control group,Al-exposed group,preventive group low-dose group, medium-dose group,and high-dose group.The mice were given food and water ad libitum.The mice were sacrificed by decapitation at the end of the treatment.The serum samples were obtained by collecting and centrafugating blood samples.The selected organs were removed.The activities of SOD,GSH-PX and the content of MDA in the blood serum and the homogenate of liver,kidney and brain tissues of the mice were determined in every group.4.2 Protecting effect of DFP on nervous system of aluminum -loaded mice 98 healthy Kunming mice,which were different in sex,were randomly divided into 7 groups by body weight;Negative control group,low-dose group,medium-dose group, high-dose rate group,Al-exposed group,preventive group and pydtinol -exposed group.the mice were given food and water ad libitum.4.2.1 The effects of DFP on learning and memory impairment caused by aluminum in mice Two days before the administration ended,step-down test was performed to investigate the effects of DFP on learning and memory impairment caused by aluminum in mice.Step-down latency(SDL)and error times in 5 minutes were noted.4.2.2 Effect of recovering the neurotransmitter in the central nervous system The activity of AchE in the blood serum and the homogenate of brain tissues of the mice were determined in every group.4.2.3 Pathomorphological observation of hippocampus T he hippocampus of the mice were fixed with 4% paraformaldehyde,and then did hematoxylin-eosin staining. Pathomorphological observations were carried out to investigate the morphological changes.4.2.4 DFP protect against the apoptosis caused by aluminum The expression of anti-apopotosis protein Bcl-2 and apopotosis protein Bax in chronic Al-exposed mice and DFP-treated hippocampus was measured by immnohistochemical method,aimed to detect if DFP could protect against the apoptosis induced by aluminum.4.3 Protective effect of DFP on damaged genetic material of mice induced by aluminum.The development of micronuclei(MN)in bone marrow cells and sperm abnormalities were used as mutagenic bioassay in Kun Ming mice.Mice were randomly divided into five groups;negative control group,cyclophosphamide positive control group,Al-exposed group,low-dose group and high-dose group.4.4 Effect of DFP on liver and renal function of aluminum-induced rats Wistar rats were administered with aluminum chloride for 3 weeks.Then they were treated with different doses of DFP.The activity of alanine aminotransferase(ALT),glutamic oxalacetic transaminase(AST),alkaline phosphatase(ALP)and the contents of total protein(TP),albumin,total bilirubin(TB)and conjugated bilirubin,creatinine(Cr), urea nitrogen(BUN)and uric acid(UA)in serum were detected.The concentrations of Al,Zn,Cu,Fe,Ca and Mg in liver and kidney tissues were measured simultaneously.Results1.The synthesis of DFPThe results showed that the melting point and the infrared spectrum and nuclear-magnetic spectrum corresponded with the structural characteristics of the target compound.DFP yield was 58.9%.2.Toxicological experiment2.1 The LD₅₀experiment of DFP Th e results showed that the LD₅₀of DFP was 562.34mg/kg.The 95% credibility range of DFP fell within 501.19～630.96mg/kg.2.2 Peripheral blood and bone marrow cell micronucleus experiment The results showed that there were no significant differences in the 1/8LD₅₀group and the 1/4LD₅₀group in comparison with the negative control group(P＞0.05);The peripheral blood and the PCE micronucleus rote of bone marrow cell of the 1/2LD₅₀group were higher than that of the negative control group(P＜0.05).2.3 Sperm aberration experiment The results showed that there were no significant differences in the 1/8LD₅₀group and the 1/4LD₅₀group in comparison with the negative control group in observations conducted in the 14th,28th,35th,42nd,and 70th day(P＞0.05); While significant differences appeared between the 1/2LD₅₀group and the negative control group in observations conducted in the 14th,28th and 35th day(P＜0.05); there were no significant differences between the 1/2LD₅₀group and the negative control group in observations conducted in the 42nd day and 70th day(P＞0.05).3.Effect of DFP removing aluminum3.1 Effect of DFP removing aluminum in mice3.1.1 Effect of DFP removing aluminum Aluminum contents of preventive group, low-dose group,medium-dose group and high-dose group in brains and kidneys is lower than that of aluminum-exposed group(P＜0.05).Aluminum contents of preventive group,low-dose group,medium -dose group is higher than that of the negative control group(P＜0.05)in brains and kidneys,yet the same condition could not be observed in high-dose group.No significant differences existed among aluminum contents of various groups in livers.As far as aluminum contents in bone be concerned,aluminum contents of preventive group,low-dose group,medium-dose group and high-dose group is lower than that of aluminum-exposed group (P＜0.05)and no significant differences exist between them and the negative control group.In comparison with aluminum contents in serum,preventive group,three doses groups are lower than aluminum-exposed group and higher than the negative control group.Dose-response relations exist among three doses groups.3.1.2 Effects of DFP removing other essential elements In comparison with magnesium contents,no significant differences existed among various groups in the livers,kidneys,brains and bones.In the contents of calcium,no significant differences existed among various groups in the livers,kidneys and bones.In livers and brains,no significant differences could be observed among zinc contents of various groups. There are no significant differences among various groups in the contents of copper contents in the livers,brains and bones.3.2 Effect of DFP removing aluminum in rabbits3.2.1 Acute removing aluminumComparison of hourly aluminum output after sterile water and DFP administration suggests that DFP could increase aluminum output above control beyond the 6h monitored after administration(P＜0.01).The peak height appeared in 2-4 hours after administration.6h cumulative excretion of aluminum in the urine rose significantly in response to DFP administration when compared to control treatment(P＜0.001).3.2.2 Combination with Vc or Mesna on urinary aluminum excretion DFP, DFP+ Vc and DFP+ Mcsna had a comparable effect on aluminum excretion,but the peak height in the aluminum excretion figure was changed.This indicates that DFP, Vc and Mcsna had a mutually interactive effect on urinary aluminum elimination.3.2.3 Comparison of the efficacy of removing aluminum with various administration of DFP Comparison of 6h cumulative excretion of aluminum in the urine,injection group could increase aluminum output above intragastrically group(P＜0.05).The peak height appeared 3 hours and 2 hours after administration for injection group and intragastrically group respectively.3.2.4 Subchronic removing aluminum DFP could highly mobilize aluminum stores from tissues.At the end of experiment the aluminum contents of bone,kidney, liver and brain in DFP groups were significantly lower than that in Al-only group. 3.2.5 Effects of DFP removing essential elements copper,zinc and manganese3.2.5.1 Acute removing aluminum Urinary excretion of copper,zinc,and manganese before and after the administration of DFP varied slightly in each group, but there was no consistent increase or decrease when all the rabbit's data were compared.3.2.5.2 Snbehronie removing aluminum Serum concentrations of copper,zinc and manganese before and after the administration of DFP varied slightly in each group,but there was no significant difference.With respect to the copper,zinc and manganese concentration,the obvious difference after DFP administration had not observed in the analyzed organs.4.Study on the mechanism of DFP protecting against chronic aluminum-exposed animals4.1 Effect of recovering the anti-oxidizing system The results showed that in comparison with the corresponding data of the model group(P＜0.05),significant differences existed in the negative control group in terms of the activities of SOD, GSH-PX and the content of MDA in liver,brain and the blood serum.In comparison with the corresponding data of the negative control group(P＞0.05),no significant differences existed among the medicine-treated groups in the terms of the activities of SOD,GSH-PX and the content of MDA in liver,kidney,brain and the blood serum.4.2 Protecting effect of DFP on nervous system of aluminum -loaded mice4.2.1 The effects of DFP on learning and memory impairment caused by aluminum in miceThe step-down test showed that the SDL of preventive group,low-dose group, medium-dose group and high-dose group is significantly lower than that of aluminium-exposed group(P＜0.01,P＜0.05).At the same time,dose-response relation exists and there is no significance between three doses groups and the negative control group.The ET of negative control group is lower than that of aluminium-exposed group(P＜0.05).There is no significance between three doses groups and the negative control group.4.2.2 Effect of recovering the neurotransmitter in the central nervous system The results showed that the activity of AchE in the model group was higher than that of the negative control group(P＜0.05).In comparison with the model group, significant differences existed in the high-dose group,medium-dose group and the preventive group(P＜0.05).No significant differences existed between the medicine-treated groups and the negative control group.4.2.3 Pathomorphological observation of the hippocampus The hippocampus damage had been found among aluminum-exposed mice in the pathological examination,significant recovering could be observed in the DFP-treated groups.4.2.4 DFP protect against the apoptosis caused by aluminum The immunohistochemical localization of Bcl-2 and Bax in the hippocampus of mice was examined.As far as Bcl-2 protein in hippocampus concerned,there is no significant differences observed in low-dose group and pyritinol-exposed group(P＞0.05)while significantly increased in medium-dose group,high-dose group and preventive group as compared with aluminium-exposed group(P＜0.05).The number of Bax positive cells of aluminium-exposed group,preventive group and low-dose group is more than that of negative control group and pyritinol-treated group(P＜0.05),and that of of the high-dose group is higher than that of pyfitinol-treated group(P＜0.05).4.3 Protective effect of DFP on damaged genetic material of mice induced by aluminum In comparison to alum treated group,the micronucleus frequency of the lower and higher dose of DFP group was apparently lower than alum treated group (p＜0.01).Significant difference could be observed between the lower and the higher DFP group(p＜0.01)and frequency of MN PCE decreased with increasing DFP doses. Of the two groups treated with DFP plus alum,the both incidences of abnormal sperms were significantly lower than that treated with alum alone(p＜0.05),yet higher than the negative control(p＜0.05)respectively.Significant difference could be observed between the lower and the higher DFP group(p＜0.05),and the incidence of abnormal sperms decreased with increasing DFP doses.4.4 Effect of DFP on liver and renal function of aluminum-induced rats4.4.1 Effect of aluminum chelating agent on liver function The result of ALT activity demonstrated that there was no statistical significance among all the groups (P＞0.05).The AST activity of low,moderate and high DFP dose groups was lower than the positive control(P＜0.01).The result of ALP activity indicated that the low DFP dose group was significantly different from the positive control(P＜0.01).The TP content of the Al group was significantly lower than the negative control(P＜0.01). The moderate and high DFP group were higher than the Al group(P＜0.01),while the same condition hasn't been exhibited between the moderate and high DFP dose groups and negative control.The result of albumin contents demonstrated that there was no statistical significance among all the groups.Compared with the negative control,the TB contents of the other 5 groups raised obviously(P＜0.05,P＜0.01).The contents of the conjugated bilirubin in all groups were not significantly different. The aluminum concentration of the three different dose groups was significantly lower than the positive control(P＜0.01).Compared with the negative control,the Zn, Cu,Fe,Ca,Mg concentrations of DFP dose groups were not significantly different (P＜0.05).4.4.2 Effect of aluminum chelating agent on renal function The BUN content of the Al group was significantly lower than the negative control.The results of UA and Cr contents demonstrated that there was no statistical significance among all the groups.The Al level in kidney remarkably rose with the increase of Al intake.The aluminum concentration of the three different dose groups was significantly lower than that of the Al group(P＜0.01).The aluminum concentration of the moderate and high DFP dose groups,as compared with the negative control,was no statistical significance.The Cu concentration of the Al group and the lower DFP dose group was significantly lower than that of the negative control,while there were no statistical significance between the moderate,high DFP dose groups and negative control. Compared with the negative control,the Zn,Fe,Ca,Mg concentrations of the lower, moderate and high DFP dose groups were not significantly different.Conclusions1.A new DFP synthesis method has been established.This method could make the synthetic time shortened and make DFP synthesis more easily and cheaper.2.DFP is a compound with low toxicity.DFP could chelated aluminum and reduces aluminum contents in animals and has no obvious effect on other essential elements such as zinc,magnesium,copper,calsium.3.DFP could improve the learning and memory ability induced by aluminum.4.DFP could counter the aluminum-induced aging of the neurotransmitter in the central nervous system.5.DFP could regulate the changes of apoptosis-related protein caused by aluminum.6.DFP has protective effect on damaged genetic material of mice induced by aluminum.7.DFP could protect liver and renal function of aluminum-induced ratsIn a word,a new DFP synthesis method has been established,the acute toxicity of DFP has been evaluated.The effect of DFP on aluminum mobilization and elimination from tissues and serum and the mechanism of DFP protecting against chronic aluminum-exposed animals has been studied first.Those studies can provide theoretical foundation for seeking a cheap,orally active and non-toxic aluminum chelator for aluminum overload.