The Effects Of Low Concentrations Of Benzene Metabolites On The HL-60 Cell Apoptosis, Proliferation, Differentiation And Its Possible Mechanisms

Benzene as an important chemical raw material is widely used in industrial and agricultural production fields. Repeated occupational benzene exposure over long periods of time may affect several hematopoietic parameters and eventually induce malignancies of the blood. Previous studies about the toxic effects of benzene focused on apoptosis and DNA damage induced by benzene and its metabolites. In real life long-term and low level of environmental pollutants was exposured. In this low level concentrations of benzene and benzene metabolites,apoptosis and DNA damage was not obvious.Whether the low concentration of benzene have harmful effects is still not clear. In this study cell model with insignificant apoptosis and survival was established and cell proliferation, cell cycle and differentiation were detected. CYP4F3, encoding LTB4 hydrolase,was found increase in the peripheral blood of benzene poisoning workers in our previous study. In this study, the expression of CYP4F3 gene and LTB4 of cell culture supernatant were detected and MAPK cell signals were also studied in this process.The finding may play important roles in the early progress of benzene poisoning.PartⅠ. The effects of low concentration of benzene metabolites on the proliferation, apoptosis and differentiation of HL-60 cellsObjective:To study the apoptosis and survival rate of human acute promyelocytic cell line HL-60 cell after different concentrations of benzene metabolites hydroquinone and phenol treatment for 96 hours; the concentration without significant effects on the apoptosis and survival was chosen to observe low concentration of hydroquinone and phenol on the HL-60 cell proliferation、cell cycle and differentiation. Materials and Methods:The AnnexinV-FITC/Propidium Iodine assays for apoptosis was used to measure cell apoptosis induced by HQ and PhOH treatment by flowcytometry.; CCK-8 detection was used to measure cell viability of HL-60 cell treatment with various concentrations of hydroquinone and phenol; PI staining by flow cytometry was used to detect cell cycle of HL-60 cell treatment with low concentration of hydroquinone and phenol; EDU fluorescence staining was used to observe the DNA replication of HL-60 cell treatment with low concentration of hydroquinone and phenol; CD11b markers was detected by flow cytometry to measure the differentiation of HL-60 cell treatment with low concentration of hydroquinone and phenol. Results:(1) Annexin V/PI double staining showed that 35μmol/l,50μmol/HQ and 10mmol/1 PhOH induced apoptosis significantly compared with the control group (P<0.05), but the low concentration of hydroquinone (15μmol/l, 25μmol/1) and phenol (100μmol/l, lmmol/1) induced apoptosis without significant difference compared with the control group. (2) CCK-8 results showed that when the HQ and the PhOH concentrations were relatively low (HQ concentration was 15μmol/1,25μmol/l, PhOH concentration was 100μmol/l, lmmol/1) cell survival rate increased, and when the HQ and the PhOH concentrations were relatively high (HQ concentration of 35μmol/l,50μmol/l, PhOH concentration of 10mmol/1) the cell viability decreased significantly compared with the control group. (3) Cell cycle analysis showed that cells from G1 phase to S phase increased significantly in the case of low concentration of HQ and PhOH compared with the control number. (4) EDU test results showed the low concentrations of HQ and PhOH induced increase of DNA replication. (5) 96 h after HQ (15μmol/l,25μmol/1) treatment of HL-60 cells, the cell surface differentiation antigen CD11b expression did not significantly increased compared to the control group; 96 h after PhOH (100μmol/1, 1mmol/1) treatment of HL-60, the cell surface differentiation antigen expression of CDllb increased significantly compared with the control group.PartⅡ. The changes of CYP4F3 gene and LTB4 and the mechanism of MAPK signal transduction by low concentrations of benzene metabolites in HL-60 cellsObjective:To observe and investigate the expression of CYP4F3 gene and the changes of LTB4 after treatment of low concentration of hydroquinone and phenol in HL-60 cells, and to explore the changes of MAPK signaling pathway in this process. Materials and methods:Fluorescence quantitative PCR was used to detect the expression of CYP4F3 gene with low concentrations of hydroquinone and phenol treatment HL-60 cells and adult peripheral blood mononuclear cells; ELIS A was used to detect the changes of LTB4 levels in the supernatant of low concentration of hydroquinone and phenol treatment in HL-60 cells; Western Blotting was used to detect the expression P38, P-P38, ERK, P-ERK protein. Results:(1) The CYP4F3 gene expression was significantly higher compared with the control after phenol (100μmol/1, 1mmol/1), all-trans retinoic acid (0.001mol/1) treatment in HL-60 cells for 96 hours and adult peripheral blood cells. (2) LTB4 in the supernatant decreased significantly after phenol (100μmol/l, lmmol/1) treatment in HL-60 cells for 96 hours but not in hydroquinone (15μmol/1,25μmol/1) treatment group. (3) P38 phosphorylation was significantly enhanced after HQ (15μmol/l,25μmol/1); PhOH (100μmol/1, 1mmol/1) treatment in HL-60 cells after 96 hours, P38 induced by LTB4 phosphorylation was inhibited by PhOH (100μmol/l, lmmol/1). (4) The ERK phosphorylation was enhanced significantly after PhOH (100μmol/1, 1mmol/1) treatment in HL-60 cells for 96 hours but not in hydroquinone (15μmol/1,25μmol/1) treatment group and the ERK phosphorylation induced by LTB4 was not affected by PhOH.Conclusions:1. Low concentrations of phenol can promote the differentiation of HL-60 cells while the low concentration of hydroquinone had no significant effect on the differentiation of HL-60 cells.2. Low concentration of phenol treatment in HL-60 cells for 96 hours induced CYP4F3 gene expression and decreased LTB4 in the supernatant significantly3. P38 phosphorylation was increased after low concentration of phenol and hydroquinone treatment in HL-60 cells for 96 hours, ERK phosphorylation was increased only by the low concentration of phenol treatment.