Study On Susceptibility And Mechanism Of Glucose-6-Phosphate Dehydrogenase Deficiency On Hematopoietic Toxicity By Benzene Exposure

Benzene is a chemical contaminant that is widespread in industrial and living environments. Benzene was a human carcinogen and long-term exposure of benzene can effect hematopoietic system, mainly due to its metabolites of phenol, hydroquinone and benzoquinone’s toxicity to bone marrow hematopoietic stem cells and hematopoietic microenvironment, leading to a variety of harmful effects including a reduction in granulocyte, bone marrow suppression, and even leukemia.Glucose-6-phosphate dehydrogenase(G6PD), the rate-limiting enzyme of the pentose phosphate pathway, converts glucose-6-phosphate(G6P) into 6-phosphogluconolactone and maintains the level of the nicotinamide dinucleotide hydrogen phosphate (NADPH), which in turn promotes the regeneration of glutathione(GSH), protecting cells against oxidative damage and injury. G6PD deficiency is the most common human enzymopathy, affecting over 400 million individuals worldwide. Persons with G6PD deficiency can experience episodes of neonatal jaundice and acute hemolysis when exposure to oxidative stresses such as certain drugs, infection or foods (particularly fava beans), although most affected individuals are asymptomatic.In the first part of this study, a mouse model of hematopoietic toxicity induced by benzene was established to investigate serum peptide biomarkers of benzene poisoned mice and control mice. Differential peptide spectra showed a two-fold increase in expression was sequenced and confirmed as G6PD. Then a stable line of human leukemia k562 cells with G6PD gene silencing in vitro was established to investigate whether inhibition of G6PD could enhance the susceptibility in K562 cells induced by benzoquinone and whether the increased oxidative damage is caused by the inhibition of GSH synthesis. Finally, G6PD low activity mice was used to study whether inhibition of G6PD could enhance the susceptibility of hematopoietic toxicity in mice induced by benzene.1. Serum peptidome analysis in mice with benzene exposureHematopoietic toxicity model of mice was established by subcutaneous injection with 150mg/(kg· d),300mg/(kg· d) of benzene. Mices were killed after the end of the exposure. General toxicity and hematopoietic toxicity of benzene to mice were evaluated on organ coefficient, peripheral blood test and the ratio of bone marrow hematopoietic stem cells. Serum peptides were processed with weak cationic exchange magnetic beads (WCX-MB) and identified by mass spectrometry using an assisted laser desorption ionization/time of flight mass spectrometry (MS). Differential peptide spectra were obtained by Nano-LC/ESI mass spectrometry and analyzed by the software BioworksBrowser 3.3.1 SP1.During the period of exposure, the renal coefficient was significantly higher and the thymus coefficient was significantly lower compared with the control group. The proportion of hematopoietic stem cells in bone marrow was significantly lower and the WBC, RBC, Hgb and Plt in the peripheral blood were significantly lower compared with the control group.Seven serum peptidome peaks showed significantly different expression between exposure groups and control group. Two peptide peaks (1231.2 and 1241.8), which showed a two-fold increase in expression, were sequenced and confirmed as G6PD and heat shock protein 90 Beta (HSP90 Beta), respectively. Furthermore, the expression of the G6PD in liver cells showed the same trend as in serum, G6PD might be the candidate serum biomarkers of benzene exposure. It also provided clues for the molecular mechanism of benzene-induced oxidative stress.2. Construction of stable k562 cells line with G6PD gene silencing in vitro to investigate the regulation of G6PD on the cytotoxicity of benzoquinone.Three siRNA lentiviruses targeted G6PD gene and a negative control siRNA lentivirus were transfected into k562 cells. RT-PCR was applied to measure the mRNA expression of G6PD. G6PD defective K562 cells(K562-G6PD△) and negative control cells were treated with 0,10,20μmol/L of benzoquinone. RT-PCR and WB were used to detect G6PD mRNA and protein expression level of K562-G6PD △ and negative control cells. Fluorescence labeled probes and colorimetric assay were used to measure the ROS and NADPH/NAdP、 GSH/GSSG level, MTT assay, comet assay, cell apoptosis assay and cell cycle assay were used to detect the proliferation inhibition, DNA damage, apoptosis rate and cycle distribution changes.The results of RT-PCR showed the G6PD mRNA expression of K562-G6PD△cells was decreased by 87% compared with negative control cells, which suggested K562-G6PD△ cells were successfully constructed. The results of RT-PCR showed the G6PD could be significantly up-regulated in K.562-G6PD △ and negative control cells exposed to benzoquinone and relative G6PD mRNA expression in K562-G6PD△ was significantly decreased compared with negative control at each concentration of benzoquinone.The protein level of G6PD showed the same trends as mRNA level in exposure and control groups. The results of colorimetric assay showed that the ratio of NADPH/NADP and GSH/GSSG in K562-G6PD△ cells were remarkably decreased compared with negative control cells at each concentration of benzoquinone. Fluorescence intensity showed the relative ROS level increased in dose-effect manner with benzoquinone exposure in K562-G6PD△ cells. The results of MTT assay indicated that the relative growth rate of K562-G6PD△ cells was remarkably decreased compared with negative control cells at each concentration of benzoquinone. Comet assay results showed tail DNA% and oliver tail moment were significantly increased at 20△mol/L benzoquinone in K562-G6PD△ cells compared with negative control cells. The apoptosis rate of K562-G6PD△ cells significantly increased compared with negative control cells at 20μmol/L group. The G2 phase of K562-G6PD△ cells was significantly increased compared with negative control cells when exposed to 10, 20μmol/L of benzoquinone.It proved that the inhibition of G6PD might not produce enough NADPH to maintain GSH when exposed to benzoquinone. Thus the relative ROS level was increased in K562-G6PD△ cells when exposed to benzoquinone. Subsequently, the DNA damage was significantly severely in K.562-G6PD△ cells, while increased apoptosis rate and G2 arrest rate were found in K562-G69D△ cells. In brief, inhibition of G6PD could enhance the oxidative damage in K562 cells induced by benzoquinone.3. Construction of G6PD low activity mice to study the regulation of G6PD on hematopoietic toxicity of benzeneFirst, the G6PD low activity mice (G6pdxa-mlNcu) was constructed. The PCR method was used to identify the mouse genotype and the colorimetric assay was used to detect G6PD enzyme activity in liver. G6pdxa-mlNeu and control mices were treated with 40 mg/(kg·d),80 mg/(kg·d),160 mg/(kg·d) of benzene. After exposure, body weight, organ coefficient, peripheral blood test and DNA damage were evaluated.PCR results showed that the mice genotypes was mution type and liver G6PD activity was only 61% compared with control mice. The spleen coefficient of G6pdxa-mlNeu was remarkably decreased compared with control mice when expose to 40,80,160mg/(kg·d) of benzene. The kidney coefficient of G6pdxa-mlNeu was remarkably decreased compared with control mice when expose to 40,80mg/(kg·d) benzene. The results of peripheral blood test and comet assay indicated that WBC was significantly decreased compared with control mice, tail DNA%, oliver tail moment of G6pdxa-mlNeu were significantly increased compared with control mice at 80 mg/(kg·d) group.The results of WBC and DNA damage suggested that G6PD low activity could enhance the susceptibility of hematopoietic toxicity and oxidative damage in mice induced by benzene.In summary, the study found G6PD was overexpression in serum peptides and liver cells in benzene-exposed mice with exhibited hematopoietic toxicities compared with normal controls, which indicated that G6PD might be involved in the toxicity of benzene exposure. The study on K562-G6PD△ cells suggested that inhibition of G6PD could inhibit GSH synthesis and enhance the oxidative toxicity susceptibility in K562 cells induced by benzoquinone. The study on G6PD low activity mice suggested that G6PD deficiency could enhance the susceptibility of hematopoietic toxicity and oxidative damage in mice.The study combinated the findings in the new study and the feasibility of theory, proposed the research of G6PD deficiency and susceptibility and mechanism of benzene hematopoietic toxicity firstly. And many countries in the world detected the activity of G6PD in neonatus, the screening of susceptible population also has a good feasibility. Therefore, the research would provide important theoretical basis of science at the risk of G6PD deficient people exposed to benzene, and may provide new biomarkers for the screening of benzene exposed susceptible population, It was of great significance to enhance the primary prevention of occupational benzene exposure.