Relationship Between GSTA1 And M1 Gene Polymorphism And GST Activity And Pharmacokinetic Interaction Between Cyclophosphamide And Busulfan Based On GST

Objective:To check GSTA1 gene polymorphism and GSTM1 gene deletion polymorphism;GST enzyme activity was measured,and the effects of GSTA1 and GSTM1 gene polymorphisms on GST enzyme activity were analyzed.The pharmacokinetics of busulfan mediated by GST enzyme was investigated in rabbits at the same time as intravenous inj ection of cyclophosphamide and busulfan.The effects of cyclophosphamide on GST activity in rabbits and HepG2 cells were investigated.The initial mechanism of cyclophosphamide-activated GST enzyme was studied.Methods:1.Detection of GSTA1 and GSTM1 gene polymorphismThe DNA in the blood was extracted by a column blood genome extraction kit,and the extracted DNA was subj ected to polymerase chain reaction by PCR-RFLP technique,and the PCR amplification product was sequenced by a DNA sequencer to determine the genotyping of GSTA1 and GSTM1.2.Determination of GST enzyme activityThe rate of increase in absorbance at 340 nm of the binding product of GSH to CDNB by the GST enzyme was measured by an ultraviolet-visible spectrophotometer to calculate the enzyme activity of GST.3.The effect of GSTA1 and GSTM1 gene polymorphism on GST enzyme activityThe results of GSTA1 and GSTM1 gene polymorphism were correlated with GST enzyme activity results,and the relationship between the two was compared by SPSS statistical software using LSD t-test test.4.Pharmacokinetic study of busulfan after concurrent administration of BUCYFive healthy rabbits were fasted for 12 hours before administration,and the doses of simultaneous injection of busulfan and cyclophosphamide in rabbits were converted according to the dose of clinical treatment,respectively.After administration,The blood samples were gathered at 0.25,0.5,0.75,1,1.5,2,and 3 h in the rabbit ear vein.After a series of treatments,the blood samples were determined by high performance liquid chromatography,and then the single-compartment model was fitted according to the blood concentration,so as to calculate the pharmacokinetic parameters of busulfan.5.Effect of cyclophosphamide on GST enzyme activity in rabbitsFive healthy rabbits were prepared,weighed and recorded separately,and fasted for 12 hours before administration.First,1 mL of blood was taken from the ear vein of 5 rabbits,and the GST activity in rabbit serum was determined by glutathione S-transferase test kit as a self-control;8 mg/kg is the dose of cyclophosphamide administered orally in clinical practice,In accordance with the conversion of human and rabbit co efficient(3.27 mg/kg),the dose of rabbits of cyclophosphamide was 26.16 mg/kg.According to the weight of each rabbit and the concentration of the drug solution prepared,calculate the volume of the drug solution that should be administered to each rabbit,and perform oral gavage.After 2 hours,take 1 mL of blood and transfer with glutathione sulfur.An enzyme test kit is used to determine serum GST enzyme activity in rabbits.6.Effect of cyclophosphamide on GST enzyme activity in HepG2 cellsThe HepG2 cells in the logarithmic growth phase were harvested,and the uniform cell suspension was evenly distributed into each new cell culture flask,and the cell concentration of each culture flask was adjusted to 5×104/mL.After 24 hours of culture,an appropriate amount of cyclophosphamide solution was added to the culture solution to make cyclophosphamide concentrations of 0,100,200,400,600,800,and 1000 μg/mL,respectively,and each concentration was made three times in parallel.After 2h,the culture solution in each culture flask was collected,and the GST enzyme activity of HepG2 cells was measured.Results:1.Detection of GSTA1 gene polymorphism According to the method of DNA sequencing,the base sequences of the four promoter regions(-631,-567,-69,-52)where the mutation may occur were directly determined,and the genotype of GSTA1 in 170 patients with hematologic malignancies was determined.The results showed that the frequency of genotype GSTA1*A*A(wild type)was 75.3%,the frequency of genotype GSTA1*A*B(heterozygous mutant)was 22.9%and the frequency of genotype GSTA1*B*B(mutation homozygous)was 1.8%.The frequencies at which the alleles GSTA1*A and*B occur were 86.8%and 13.2%.It was found by chi-square test that there was no significant difference in GSTA1 genotype distribution between different genders(p=0.743),and it was considered that there was no difference in GSTA1 genotype distribution between male and female,which was consistent with Hardy-Weinberg equilibrium.2.Detetction of GSTA1 gene polymorphismAccording to the DNA sequencing results of the PCR products,the deletion polymorphism of the GSTM1 gene in 170 patients with hematological diseases was established.A total of 61 patients had a deletion of the GSTM1 gene fragment,109 patients had a complete GSTM1 gene sequence,and the overall deletion rate of GSTM1 gene was 35.9%.Of the 93 male patients,33 had GSTM1 gene deletion,and the deletion rate was 35.5%;29 of 77 female patients had GSTM1 deletion,and the deletion rate was 37.7%.Chi-square test showed that there was no significant difference in the deletion rate of GSTM1 gene under different genders(p=0.796),and it can be considered that there was no difference in the GSTM1 gene deletion rate between men and women,in line with Hardy-Weinberg equilibrium.3.Determination of GST enzyme activityGST enzyme activity was determined in 170 patients with hematological malignancies according to the glutathione S-transferase kit.The results showed that the GST enzyme activity of these 170 samples was subject to a positive skew distribution.The average GST activity of the male group was 5.20±0.13nmol/min/mL,and the average enzyme activity of the female group was 5.17±0.12nmol/min/mL.After t-test,there was no difference in the distribution of GST enzyme activity between different sexes.Divided into four groups by 11-30,31-50,51-70 and 71-90,the GST enzyme activities were 5.24±0.25 nmol/min/mL,5.09±0.17 nmol/min/mL,5.18±0.13 nmol/min and 5.32±0.27 nmol/min/mL,separately.The least significant difference t-test(LSD t-test)was used to detect differences among groups.With all the p values were more than 0.05,the difference in GST activity between different age groups was not significant.It can be considered that there is no difference in the distribution of GST enzyme activity between different ages.4.The effect of GSTA1 gene polymorphism on GST enzyme activityThe average enzymatic activity of wild type GSTA1 A/A was 5.34±1.26nmol/min/mL,the average enzymatic activity of heterozygous mutant GSTA1 A/B was 4.83±0.76nmol/min/mL and the average enzyme activity hom ozygous mutant GSTA1 B/B was 3.23±0.07 nmol/min/mL.Through LSD t-test test,the comparison between different groups showed that all p values were less than 0.05.It can be considered that there are differences in GST enzyme activities between different genotypes of GSTA1,that is,the order of enzyme activity of different genotypes is:wild type>heterozygous mutant>homozygous mutant.5.The effect of GSTM1 gene polymorphism on GST enzyme activityGST activity and GSTM1 gene deletion polymorphism in 170 samples were analyzed.The results revealed that the average GST activity was 5.09±1.24 nmol/min/mL in 62 GSTMI gene deletion populations,and 5.23±1.17 nmol/min/mL in 108 GSTM1 gene intact populations.By the method of t/test,p=0.424>0.05,there was no difference in GST enzyme activity between GSTMI gene deletion type(-)and non-deletion type(+),that is,the deletion of GSTM1 gene did not significantly affect GST enzyme activity.6.Pharmacokinetic study of busulfan after concurrent administration of BUCYA single-compartment model was fitted to the blood concentration of busulfan in rabbits to calculate the pharmacokinetic parameters of busulfan in 5 rabbits:half-life t1/2=1.46±0.26(h),AUC=8.097±0.795(h*mg/L),initial concentration C0=3.907±0.472(mg/L).The pharmacokinetic parameters of intravenous injection of busulfan were compared:the half-life t1/2=1.05±0.153(h),the area under the curve AUC=3.846±0.213(h*mg/L),the initial concentration C0=2.463±0.245(mg/L).The results showed that after intravenous injection of cyclophosphamide and busulfan,the elimination half-life tin of the white rabbit in rabbits became longer,and the initial concentration C0 and the area under the curve were larger.7.Effect of cyclophosphamide on GST enzyme activity in rabbitsThe average GST enzyme activities of the five rabbits before and after cyclophosphamide pretreatment were calculated to be 6.785±0.9799 nmol/min/mL and 13.1468±3.3256 nmol/min/mL,respectively.By the method of paired t-test,p=0.012<0.05,that is,the GST enzyme activity in rabbits was different before and after cyclophosphamide treatment.It can be considered that the activity of GST enzyme in rabbits was enhanced after cyclophosphamide pretreatment.8.Effect of cyclophosphamide on GST enzyme activity in HepG2 cellsWhen the concentration of cyclophosphamide was 0,100,200,400,600,800 and 1000 μg/mL,the corresponding GST enzyme activities of HepG2 cells were 2.266±0.050,2.530±1.174,3.458±0.187,3.622±0.012,3.691±0.106,3.830±0.116 and 3.941±0.065 nmol/min/mL,separately.When the concentration of cyclophosphamide was in the range of 0-200 μg/mL,the activity of GST enzyme in HepG2 cells increased significantly.When the concentration of cyclophosphamide was more than 200 μg/mL,the activity of GST enzyme in HepG2 cells was basically unchanged.Conclusion:1.Through PCR-RFLP reaction combined with DNA sequencing,the genetic polymorph isms of GSTA1 and GSTM1 in 170 patients with hematologic malignancies were accurately,intuitively and reliably studied.GST enzymes in 170 patients with hernatologic malignancies were also determined.Activity,the order of enzyme activity of different genotypes of GSTA1 was found to be:wild type(GSTA1 A/A)>heterozygous mutant(GSTA1 A/B)>homozygous mutant(GSTA1 B/B),GSTM1 gene deletion type and There was no difference in the activity of the undeleted GST enzyme,indicating that the GSTM1 genotype is not the main factor affecting the activity of GST enzyme,while the GSTA1 genotype dominates the GST enzyme activity,and the GST1 wild type corresponds to the strongest GST enzyme activity,and the GSTA1 heterozygous mutant The corresponding GST enzyme activity is second,and the GSTA1 homozygous mutant corresponds to the lowest GST enzyme activity.This provides important theoretical support for the design of clinical individualized drug delivery,helping to achieve better precision treatment.2.After pretreatment with cyclophosphamide in rabbit,the elimination half-life of busine in rabbits became shorter(t1/2 before=1.363±0.24 h,t1/2 after=0.867±0.167 h,p=0.016),indicating After cyclophosphamide pretreatment,the metabolism of busulfan in rabbits accelerated,and this study compared GST activity in rabbits and HepG2 cells before and after cyclophosphamide pretreatment,and found that GST activity increased,indicating that GST can be activated by cyclophosphamide,which also confirms the accelerated metabolism of busulfan in rabbits.However,after intravenous injection of cyclophosphamide and busulfan in rabbits,it was found that the elimination half-life of busulfan became longer(t1/2BU=1.05±1.153h,t1/2BUCY=1.46±0.26h,p=0.047),which is indicated that cyclophosphamide and busulfan will compete with GST at the time of simultaneous administration,resulting in slowing down of the metabolism of busulfan.This provides an important theoretical basis for the design of the clinical BUCY dosing schedule,which can help achieve better precision treatment.