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Objective:To investigate the effect of sodium arsenite (NaAsO 2) on transcriptional activity of nuclear factor E2-related factor 2 (Nrf2) signaling pathway in mouse lymph node vascular endothelial cell line (SVEC4-10). Methods:In vitro cell culture method was used to treat SVEC4-10 cells for 24 h with different doses of NaAsO 2 [0 (control), 2, 5, 10, 20, 50, 100, 150 μmol/L], and the cell viability was detected by tetrazole compound (MTS) method. The time-response relationship was studied with SVEC4-10 cells treated with 5 μmol/L NaAsO 2 for 0 (control), 2, 6 and 12 h; the dose-response relationship was studied with SVEC4-10 cells treated with 0 (control), 2, 5 and 10 μmol/L NaAsO 2 for 6 h; real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA expression of Nrf2 and its downstream genes glutamate-cysteine ligase catalytic subunit (Gclc), glutamate-cysteine ligase modifier subunit (Gclm), NAD(P)H dehydrogenase quinone 1 (Nqo1) and metallothionein 1 (Mt1). Establishment of Nrf2 gene stably silenced (Nrf2-KD) cells using SVEC4-10 cells, the interference control (scramble, SCR) cells and Nrf2-KD cells were treated with 0(control), 10 and 20 μmol/L NaAsO 2 for 16 h, and apoptosis was detected by flow cytometry. Results:MTS test results showed that the cell viability of the control, 2, 5, 10, 20, 50, 100, 150 μmol/L NaAsO 2 treatment groups was (100.00 ± 19.53)%, (98.18 ± 9.85)%, (96.09 ± 30.04)%, (90.64 ± 8.74)%, (59.75 ± 12.09)%, (35.43 ± 8.58)%, (26.35 ± 5.89)% and (17.54 ± 4.48)%, respectivily. There was statistically significant difference in cell viability between different dose groups ( F = 18.30, P < 0.05); and the cell viability of the 20, 50, 100, 150 μmol/L NaAsO 2 treatment groups was significantly lower than that of the control group ( P < 0.05). The time-response relationship results showed that there were statistically significant differences in Nrf2, Gclc, Gclm, Nqo1 and Mt1 mRNA level between control, 2, 6 and 12 h treatment groups ( F = 56.69, 85.28, 90.82, 80.46, 758.60, P < 0.05); with extension of arsenic exposure time, the mRNA level of Nrf2, Gclc, Gclm and Mt1 first increased and then decreased, the mRNA level of Nqo1 increased continually; among them, the mRNA level of Nrf2 peaked at 2 h, the mRNA levels of Gclc, Gclm and Mt1 peaked at 6 h, and the mRNA level of Nqo1 peaked at 12 h. The dose-response relationship results showed that there were statistically significant differences in Nrf2, Gclc, Gclm, Nqo1 and Mt1 mRNA levels between control, 2, 5 and 10 μmol/L NaAsO 2 treatment groups ( F = 68.39, 72.26, 30.41, 397.00, 28.88, P < 0.05); with increasing of arsenic exposure dose, the mRNA levels of Nrf2, Gclc, Gclm, Nqo1 and Mt1 increased. The mRNA level of Nrf2 peaked at a dose of 5 μmol/L, and the mRNA levels of Gclc, Gclm, Nqo1 and Mt1 peaked at a dose of 10 μmol/L. Apoptosis test results showed that there were statistically significant differences in the apoptosis rates of SCR and Nrf2-KD cells between control, 10 and 20 μmol/L NaAsO 2 treatment groups ( F = 8.18, 9.66, P < 0.05); compared with the control group, the apoptosis rates of SCR and Nrf2-KD cells in the 20 μmol/L NaAsO 2 treatment group increased ( P < 0.05); and the apoptosis rate of Nrf2-KD cells in the 20 μmol/L NaAsO 2 treatment group was higher than that of SCR cells in the same dose group ( P < 0.05). Conclusions:NaAsO 2 exposure has caused the activation of Nrf2 signaling pathway in mouse lymph node vascular endothelial cell line SVEC4-10 cells, activated the adaptive antioxidant response, and altered transcriptional activity; while silence of Nrf2 has made SVEC4-10 cells more sensitive to NaAsO 2 toxicity.
ABSTRACT
Inorganic arsenic (iAs) is a metalloid element that exists widely in the environment. Chronic iAs exposure may result in various types of cancer and chronic diseases. Although the mechanism of iAs toxicity is complicated, oxidative stress is the most crucial one that has been verified. Accumulating domestic and foreign epidemiological evidence shows that chronic iAs exposure through drinking water is strongly correlated with the prevalence of type 2 diabetes mellitus (T2DM). Mechanistic studies display that iAs exposure causes pancreaticβ-cell dysfunction and insulin resistance. The dynamic balance between reactive oxygen species (ROS) production and antioxidant response is one of the key factors maintaining normal physiological function. ROS plays a significant role in regulating glucose-stimulated insulin secretion and insulin signal transduction. This paper reviews the role of adaptive antioxidant response mediated by nuclear factor E2-related factor 2 ( Nrf2 ) in ROS signaling disorder resulted from chronic iAs exposure, and ROS signaling in the physiological function of pancreatic β-cell.
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Objective To explore the effects of chronic sodium arsenic (NaAsO2) exposure on energy metabolism in human keratinocytes (HaCaT).Methods Malignant transformation was induced when HaCaT cells were continuously exposed to an environmentally relevant level (0.1 μ mol/L) of inorganic arsenic for 28 weeks,HaCaT and malignantly transformed cells were kept in Group of Chronic Disease and Environmental Genomics,School of Public Health,China Medical University.Basal oxygen consumption rate and extracellular acidification rate in control group and malignantly transformed group were measured by Agilent Seahorse XF24.Changes of glucose intake in malignantly transformed HaCaT cells were detected by 2-NBDG.Cells in logarithmic growth phase were collect,mRNA levels of key components in glycolytic pathwa [glucose-6-phosphate dehydrogenase (G6PD),phosphofructokinase (PFKL),glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] and mitochondrial respiratory chain complex [NADH dehydrogenase iron-sulfur protein subunit 3 (NDUFS3),succinate dehydrogenase iron-sulfoprotein subunit (SDHB),cytochrome C (CYTC),CYTC oxidase subunit Ⅳ (COX4L1),ATP synthase F1 subunit (ATP5B),and ATP synthase F0 subunit (ATP5G1)] were determined by real-time quantitative PCR (RT-qPCR).Results The basal oxygen consumption rate of malignantly transformed group [(44.784 ± 10.159) pMoles/min] was lower than that of control group [(66.842 ± 15.756) pMoles/min,t =4.914,P ＜ 0.05].The glucose intakes of malignantly transformed group (1.592 ± 0.410 for cells cultured without serum,and 1.631 ± 0.323 for cells cultured with serum) were significantly higher than those of control group (1.000 ± 0.334,1.000 ± 0.196,t =2.916,4.068,P ＜0.05).The mRNA levels of G6PD (0.555 ± 0.009),NDUFS3 (0.623 ± 0.031) and SDHB (0.702 ± 0.094)in malignantly transformed group were significantly lower than those of control group (1.000 ± 0.027,1.000 ±0.034,1.000 ± 0.114,t =23.690,11.340,2.814,P ＜ 0.05);the mRNA levels of PFKL (1.787 ± 0.176),GAPDH (1.466 ± 0.111),CYTC (2.461 ± 0.179),ATP5B (1.956 ± 0.161),and ATP5G1 (2.055 ± 0.052) were significantly higher than those of control group(1.000 ± 0.153,1.000 ± 0.069,1.000 ± 0.030,1.000 ± 0.091,1.000 ± 0.237,t =4.696,4.985,11.540,7.436,6.021,P ＜ 0.05).Conclusion When continuously exposed to an environmentally relevant level of inorganic arsenic,the glucose metabolic pattern of HaCaT cells is changed along with lower oxygen consumption and higher glycolysis levels.
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Objective To study the effects of chronic exposure to inorganic arsenic (iAs) in drinking water on bone mineral density (BMD) in mice and its underlying mechanisms.Methods Five-month-old female C57BL/6 mice were randomly divided into sham groups and ovarectomy (OVX) groups (n =19 mice each group),which were further randomly assigned into control group (distilled water) and iAs exposure groups [5 mg/L and 20 mg/L,inorganic arsenite (iAsⅢ):inorganic arsenate (iAsv) =1 ∶ 1].Following 3 months of exposure to iAs,BMD of the mice were determined by the dual energy X-ray detector.RAW 264.7 cell line and bone marrow hematopoietic stem cells (BMHSC) primarily isolated from C57BL/6 mice were used to study the in vitro effects of iAs on osteoclast differentiation and underlying mechanisms.During differentiation induced by receptor activator of nuclear factor-κ B ligand (RANKL,50 μg/L) and macrophage colony-stimulating factor (M-CSF,30 μg/L),RAW 264.7 cell line were treated with 0.00,0.25,0.50,0.75,1.00,1.50 μmol/L iAsⅢ,while BMHSC with 0.0,0.2,0.4,0.6,0.8,1.0 μmol/L iAsⅢ for 6 days.Based on the effect of iAsⅢ on the differentiation of RAW cells,RAW 264.7 cell line were treated by 0.6 μmol/L iAsⅢ combined with 0,5,10 mmol/L of N-acetyl-cysteine (NAC).Tartrate resistant acid phosphatase (TRAP)-positive red-colored cells with 3 or more nuclei were considered mature osteoclast.Results The femoral BMD of the mice [(80.04 ± 4.06) mg/cm2] that had been exposed to 20 mg/L of iAs for 3 months was substantially decreased compared to that of sham control mice [(84.44 ± 4.40) mg/cm2].As expected,the BMD of the OVX group [(76.36 ± 3.36) mg/cm2] was significant decreased compared to that of the sham control group (P ＜ 0.05).However,the BMD among the OVX groups showed no significant difference [5 mg/L:(77.74 ± 4.91) mg/cm2;20 mg/L:(75.56 ± 3.71) mg/cm2,P ＞ 0.05].In vitro studies,the iAsⅢ evidently affected the osteoclast differentiation in a concentration-dependent fashion.Low concentrations of iAs Ⅲ exposure significantly augmented osteoclast differentiation in the two cell models while high concentrations showed inhibitory effect.In RAW 264.7 cells,the number of osteoclasts in different groups was significantly different (F =1 522,P ＜ 0.05),in the 0.50 μmol/L iAs Ⅲ group the number of osteoclasts reached the peak.In the BMHSC,the nmnber of osteoclasts in different groups was also significantly different (F =1 781,P ＜ 0.05),in the 0.6 μmol/L iAsⅢ group the number of osteoclasts reached the peak.NAC pretreatment significantly abolished low-level iAsⅢ(0.6 μmol/L)-induced augmentation of osteoclast differentiation in a concentration-dependent fashion (0 mmol/L:109.33 ± 3.06;5 mmol/L:56.00 ± 2.65;10 mmol/L:22.67 ± 0.58,F =1 940,P ＜ 0.05).Conclusions The inhibitory effect of iAs on bone metabolism is dependent on the availability of ovary function,suggesting that iAs may interfere with estrogen metabolism and/or function to disturb bone metabolism.Oxidative stress induced by iAs exposure stimulates osteoclast differentiation,and the increased osteoclast differentiation may be involved in the reduction of BMD caused by chronic iAs exposure.These preliminary findings suggest that antioxidant intervention may be an effective approach to prevent osteoporosis induced by chronic iAs exposure.