Metabolic Characteristics Of G6PD Low Expression Mice And The Effect Of Benzene Exposure On IT

The pentose phosphate pathway is the first step in glycolysis,glucose-6-phosphate dehydrogenase（G6PD）is the key enzyme in the glucose pentose phosphate metabolism pathway.G6PD can catalyze the oxidative dehydrogenation of glucose-6-phosphate to produce NADPH,which is the only way to produce NADPH in red blood cells.NADPH can maintain glutathione（GSH）in a restore state,glutathione in a restore state can balance the oxidative damage caused by superoxide and protect the body from oxidative damage.Therefore,G6PD is very important in the antioxidant process.The low expression of G6PD can cause insufficient production of NADPH and the decrease of GSH content in cells,thus reducing the body’s ability to deal with oxidative damage and being vulnerable to external oxidative damage.G6PD deficiency is a genetic disease affecting hundreds of millions of people.It has a high incidence in southern China,such as Guangdong,Hainan,and Guangxi.Because the low expression of G6PD is a genetic disease,and the G6PD gene is located on X chromosome,it is difficult to diagnose and treat G6PD deficiency.At present,there is no good way to deal with this disease in clinic.Benzene is a human carcinogen.Many studies have found that the toxicity of benzene is closely related to its metabolism.The metabolites of benzene play an important role in benzene-induced diseases.In the previous study of our research group,we found that when exposed to benzoquinone,the ratio of NADPH/NADP and GSH/GSSG in G6PD low expression cells was lower than that in the control cells,and the level of ROS in cells was significantly higher than that in the control cells.In animal experiments,it was found that benzene exposure had more significant hemopoietic toxicity on G6PD low expression mice than that of C3H mice.The low expression of G6PD may not produce enough NADPH to maintain the restore state of glutathione,resulting in the increased ROS,increased DNA damage,increased apoptosis and other oxidative damage,however,the mechanism of the low expression of G6PD on benzene toxicity is not completely clear.Metabonomics is a new omics technology.It is an important part of system biology.At present,it is mainly used for early diagnosis of diseases,the discovery of drug targets and the search for specific biomarkers.The non-targeted metabolomics is a technique for qualitative and semi-quantitative research on metabolites in biological fluids to understand the changes in the expression of metabolites.In this study,the metabolic characteristics of bone marrow cells,plasma and urine of G6PD low expression mice,C3H mice,benzene exposed G6PD low expression mice and benzene exposed C3H mice were analyzed by high performance liquid chromatography-time-of-flight mass spectrometer to screen the potential specific small molecule metabolites in bone marrow cells,plasma and urine,analyze the possible metabolic pathways involved,so as to provide theoretical basis and clues for exploring the potential biomarkers of G6PD low expression and the effect of G6PD low expression on benzene exposed mice,and provide new ideas for the diagnosis and prevention of G6PD low expression.1.Establishment of G6PD low expression mice and benzene exposed mice modelsEstablish C3H mice with low G6PD expression(G6pd^a-m1Neu/Y)and C3H mice with normal G6PD expression.The G6PD low expression mice and C3H mice were exposed to benzene（150 mg/kg·d）by subcutaneous injection with grain blending oil as solvent to establish the benzene-exposed animal models.The G6PD low expression mice and C3H mice in the control group were established by subcutaneous injection of grain blending oil.After the establishment of the model,the body weight,organ coefficient,peripheral blood routine and urine S-phenylmercapturic acid（S-PMA）concentration were dectected.The results showed that the weight of mice in the four groups increased without statistical difference.In the blood routine analysis,the white blood cell and red blood cell counts of G6PD low expression mice exposed to benzene were lower than those of the control group（P<0.05）,and the white blood cell counts of C3H mice exposed to benzene were lower than those of C3H mice in the control group（P<0.05）.In benzene exposed group,the leukocyte and platelet count of G6PD low expression mice exposed to benzene were lower than those of C3H mice（P<0.05）,and there was no significant difference in peripheral blood cell counts of other groups（P>0.05）.The kidney body ratio of G6PD low expression mice was lower than that of C3H mice（P<0.05）,the spleen body ratio of benzene exposed G6PD low expression mice and benzene exposed C3H mice were lower than that of the control group（P<0.05）.S-PMA was detected in the urine of the 4 groups of mice,and the results showed that the urine S-PMA level of benzene exposed G6PD low expression mice and benzene exposed C3H mouse urine was higher than that of the control group G6PD low expression mice and control group C3H Mice（p<0.05）.2.Analysis of the effect of low expression of G6PD on the metabolic characteristics of miceThe plasma,urine and bone marrow cells of G6PD low expression mice and C3H mice were collected,detected by high-performance liquid chromatography time-of-flight mass spectrometry,using principal component analysis and orthogonal partial least square analysis,combined with the human metabonomics database,Kyoto gene and genome encyclopedia,and metaboanalyst 4.0,a platform for quantitative metabonomics data,to screen,identify and analysis the differential metabolites in bone marrow cells,plasma and urine,and to analyze the possible metabolic pathways.The results showed that in G6PD low-expression mice and C3H mice,a total of 2028substances with VIP value>1 and p<0.05 were screened in positive and negative ion mode,and 45 potential differential metabolites were identified.A total of 20 differential metabolites were screened in bone marrow cells,of which the level of 8 metabolites increased and the level of 12 metabolites decreased.A total of 17 differential metabolites were screened in plasma,of which 12 metabolites increased and the level of 5 metabolite decreased.A total of 8 differential metabolites were screened in urine,of which the level of 6 metabolites increased and the level of 2 metabolites decreased.The metabolic pathways that may be affected by the low expression of G6PD include phenylalanine,tyrosine and tryptophan biosynthesis pathway,phenylalanine metabolism pathway,purine metabolism pathway,α-linolenic acid metabolism pathway,glycerol phospholipid metabolism pathway and histidine metabolism pathway.3.Analysis of the effect of low expression of G6PD on the metabolic characteristics of benzene exposed miceThe plasma,urine and bone marrow cells of benzene exposed G6PD low-expression mice and benzene exposed C3H mice were collected,detected by high-performance liquid chromatography time-of-flight mass spectrometry,using principal component analysis and orthogonal partial least square analysis,combined with the human metabonomics database,Kyoto gene and genome encyclopedia,and metaboanalyst 4.0,a platform for quantitative metabonomics data,to screen,identify and analysis the differential metabolites in bone marrow cells,plasma and urine,and to analyze the possible metabolic pathways.The results showed that in benzene exposed G6PD low-expression mice and benzene exposed C3H mice,a total of 2997 substances with VIP value>1 and p<0.05 were screened in positive and negative ion mode,and 21 potential differential metabolites were identified.A total of 7 differential metabolites were screened in bone marrow cells,of which the level of 4metabolites increased and the level of 3 metabolites decreased.A total of 9 differential metabolites were screened in plasma,of which the level of 9 metabolite decreased.A total of 5differential metabolites were screened in urine,of which the level of 4 metabolites increased and the level of 1 metabolites decreased.The low expression of G6PD may affect purine metabolism pathway,a-linolenic acid metabolism pathway and taurine and hypotaurine metabolism pathway in benzene exposed mice.conclusionIn this study,animal models of G6PD low expression mice,benzene exposed G6PD low expression mice and benzene exposed C3H mice were successfully established.Bone marrow cells,plasma and urine metabonomics of G6PD low expression mice,C3H mice,benzene exposed G6PD low expression mice and benzene exposed C3H mice were analyzed.The results showed that the low expression of G6PD affected phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,purine metabolism,α-linolenic acid metabolism,glycerophospholipin metabolism and histidine metabolism.The low expression of G6PD could change purine metabolism pathway,α-linolenic acid metabolism pathway and taurine and hypotaurine metabolism pathway in benzene exposed mice.