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BackgroundArsenic exposure is a common and important environmental and occupational hazardous factor in China, and arsenic-induced insulin resistance (IR) has attracted widespread attention as a negative health outcome to the population. ObjectiveTo explore part of the mechanism of hepatic IR induced by arsenic exposure based on the peroxisome proliferators-activated receptors γ (PPARγ)/ glucose transporter 4 (GLUT4) pathway, and to investigate potential effects of Ginkgo biloba extract (GBE) on hepatic IR induced by arsenic exposure and associated mechanism of action. MethodsThe target of drug action was predicted by network pharmacology and verified by in vivo and in vitro experiments. In vivo experiments: 48 SPF C57BL/6J male mice were divided into 4 groups, including control group, 50 mg·L−1 NaAsO2 model group (NaAsO2), 50 mg·L−1 NaAsO2+10 mg·kg−1 GBE intervene group (NaAsO2+GBE), and 10 mg·kg−1 GBE group (GBE), 12 mice in each group. The animals were given free access to purified water containing 50 mg·L−1 NaAsO2, or given intraperitoneal injection of normal saline containing 10 mg·kg−1 GBE once per week. After 6 months of exposure, blood glucose detection, intraperitoneal glucose tolerance test (IPGTT), and insulin tolerance test (ITT) were performed. Serum and liver tissues were collected after the mice were neutralized, liver histopathological sections were obtained, serum insulin levels, liver tissue glycogen content, glucose content were detected by enzyme linked immunosorbent assay (ELISA), and the expression of PPARγ and GLUT4 proteins was detected by Western blot (WB). In vitro experiments: HepG2 cells were divided into 4 groups, including control group, 8 μmol·L−1 NaAsO2 group (NaAsO2), 8 μmol·L−1 NaAsO2 + 200 mg·L−1 GBE intervene group (NaAsO2+GBE), and 200 mg·L−1 GBE group (GBE). The levels of glycogen and glucose were detected by ELISA, and the expression of PPARγ and GLUT4 proteins was detected by WB. Results A strong binding effect between GBE and PPARγ was revealed by network pharmacology. In in vivo experiments, the NaAsO2 group exhibited an elevated blood glucose compared to the control group, and the NaAsO2+GBE group showed a decreased blood glucose compared to the NaAsO2 group (P

BackgroundArsenic exposure can lead to liver dysfunction, liver steatosis, hepatitis, liver fibrosis, and other liver injuries, but its mechanism is still unclear and effective treatment methods are lacking.ObjectiveTo investigate the potential intervention effect and associated mechanism of Rosa roxburghii Tratt juice on arsenic-induced liver injury in rats from the perspective of S-adenosylmethionine (SAM) metabolism.MethodsThirty-six Wistar rats were randomly divided into six groups, six rats in each group, half male and half female. The low, medium, and high dose groups of sodium arsenite (NaAsO2) were given 2.5, 5.0, and 10.0 NaAsO2 mg·kg−1 (body weight, thereafter), respectively; the control group was given 10 mL·kg−1 deionized water; the Rosa roxburghii Tratt juice intervention group was given 10 mg·kg−1 NaAsO2 for 4 h and then 10 mL·kg−1 Rosa roxburghii Tratt juice; the Rosa roxburghii Tratt juice control group was given 10 mL·kg−1 Rosa roxburghii Tratt juice by gavage. All rats were gavaged once a day, 6 d per week for 4 months. Histopathological changes of rat liver were observed by hematoxylin-eosin (HE) staining. Total bile acids (TBA) and γ-glutamyl transpeptidase (γ-GT) were detected by enzyme cycle method and rate method, respectively. Ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS) was used to evaluate SAM and S-adenosylhomo cysteine (SAH) in rat liver. Real-time fluorescence quantitative reverse transcriptional PCR (RT-qPCR) was used to detect the mRNA expression levels of the genes of glycine-N-methyltransferase (Gnmt), nicotinamide-N-methyltransferase (Nnmt), and phosphatidylethano-lamine N-mathyltransferase (Pemt).ResultsIn the control group, the liver nuclei were stained clearly, the cytoplasm was stained uniformly, and the liver cords were arranged radially without inflammatory infiltration. In the low, medium, and high dose groups of NaAsO2, hepatic sinusoidal dilation, congestion, and inflammatory infiltration were observed to different degrees. Compared with the control group, TBA in the medium and high dose groups of NaAsO2, and γ-GT in the high dose group of NaAsO2 were significantly increased (P

BackgroundThe accumulation of inorganic arsenic in the liver can lead to liver fibrosis. Solute carrier family 7 member 11 (SLC7A11) and cyclin dependent kinase inhibitor 1A (CDKN1A) are ferroptosis related genes that are abnormally expressed in liver and fibrosis diseases, but their impacts on inorganic arsenic induced liver fibrosis has not been reported yet. ObjectiveTo explore the effect of different doses of sodium arsenite on the migration ability of human hepatic stellate (LX-2) cells and the changes in DNA methylation and expression of SLC7A11 and CDKN1A in LX-2 cell activation induced by sodium arsenite. MethodsLX-2 cells were treated with different doses of sodium arsenite for 24 h. The experiment set up four groups: control group (0 μmol·L−1), low-dose group (5 μmol·L−1), medium-dose group (10 μmol·L−1), and high-dose group (15 μm·L−1). Possible morphological changes of LX-2 cells were observed under an inverted microscope in each group. Cell migration and movement were determined by cell scratch assay. Mitochondrial morphology was observed in the control and the high-dose groups under a transmission electron microscope (TEM). Fe2+ fluorescence intensity was detected with a FerroOrange fluorescence probe. Differentially methylated sites were screened using the Illumina 850 K methylation chip. Ferroptosis related markers (SLC7A11 & CDKN1A) and hepatic stellate cell activation related markers [transforming growth factor-β1 (TGF-β1) and collagen type Ⅲ (Collagen Ⅲ)] were detected by real-time fluorescence quantitative PCR and Western blot. ResultsWith the increase of exposure dose, the cells retracted tentacles and gradually became round, and the healing of cell scratches gradually deteriorated. Destruction of mitochondrial membrane integrity and mitochondrial crista fracture in some cells were observed under a TEM in the high-dose group. Laser confocal microscopy showed that the fluorescence intensity of Fe2+ gradually increased with the increase of exposure dose. The results of methylation chip displayed that compared with the control group, high methylation was found in the high-dose group at the cg22659014 site in the promoter region of SLC7A11, and at the cg17964532 site in the promoter region of CDKN1A. With the increase of exposure dose, the mRNA and protein expression levels of SLC7A11 decreased (P0.05), and its protein expression levels in the low-, medium-, and high-dose groups were higher than that in the control group (P

BACKGROUND: Vascular endothelial cells are the main target of arsenic toxicity and the molecular mechanism of arsenic-induced endothelial cell injury needsto be further studied. OBJECTIVE: To study the effects of sodium arsenite on human umbilical vein endothelial cell injury and sphingosine kinase 1/sphingosine 1-phosphate signaling axis of human umbilical vein endothelial cells. METHODS: Human umbilical vein endothelial cells were isolated, cultured, and identified. The cells were treated with 0, 5, 10, 15, 20, 25 µmol/L sodium arsenite for 24 hours. Cell viability was detected by cell counting kit-8. Cell morphology was observed by inverted phase contrast microscope. The content of intracellular reactive oxygen species was detected by fluorescent probe DCFH-DA. Annexin V-FITC/PI double labeling combined with flow cytometry and TUNEL were used to detect apoptosis. The content of sphingosine 1-phosphate in cells was detected by ELISA. The mRNA and protein expressions of vascular cell adhesion molecule-1, sphingosine kinase 1, and sphingosine 1-phosphate were detected by real-time fluorescence quantitative PCR and western blot respectively. RESULTS AND CONCLUSION: Compared with the control group (0 µmol/L sodium arsenite), the cell viability decreased gradually along with the increased concentration of sodium arsenite in a dose-dependent manner; the cell fusion rate decreased gradually, the cell gap widened gradually, and the number of exfoliated and floating cells increased gradually; the reactive oxygen species content and apoptosis rate increased gradually; the sphingosine 1-phosphate content in cells increased gradually; the relative mRNA and protein expressions of vascular cell adhesion molecule-1 and sphingosine kinase 1 increased gradually, but the relative mRNA and protein expression of sphingosine kinase 1 decreased gradually. To conclude, sodium arsenite-injured human umbilical vein endothelial cell injury may be related to the activation of sphingosine kinase 1/sphingosine 1-phosphate signaling axis and the downregulation of sphingosine 1-phosphate receptor 1. Sphingosine kinase 1/sphingosine 1-phosphate signaling axis may become a new target of arsenic-induced cardiovascular injury. [ABSTRACT FROM AUTHOR]

Objective To observe the effect of sodium arsenite (NaAsO2) on the activation of human hepatic stellate cells and its relationship with ferroptosis. Methods Human liver stellate cells LX-2 were cultured in vitro, and were divided in the NaAsO2 group and control group. Cells in the NaAsO2 group were treated with 5, 10 and 15 µmol/L NaAsO2 for 24 h, respectively, and cells in the control group underwent normal culture. The morphological changes of cells were observed under inverted microscope. The content of glutathione (GSH) was determined by colorimetry. Fluorescent probe was used to detect cell lipid reactive oxygen species (ROS). Ferroptosis markers SLC7A11 and glutathione peroxidase 4 (GPX4), fibrosis markers α smooth muscle actin (α-SMA) and CollagenⅠmRNA were detected by RT-PCR. Spearman correlation analysis was used to analyze the correlation between fibrosis index and ferroptosis index. Results With the in‑ crease of NaAsO2 concentration, the cell antennae gradually retreated, the number of cells decreased, and the intercellular space expanded. Compared with the control group, the GSH content of cells in different NaAsO2 concentration groups decreased, and the GSH content in the 15µmol/L NaAsO2 group was lower than that in the 10 and 5 µmol/L NaAsO2 groups, and that in the 10 µmol/L NaAsO2 group was lower than that in the 5 µmol/L NaAsO2 group (all P<0. 05). Compared with the control group, the fluorescence intensity of lipid ROS in the NaAsO2 groups was enhanced. Compared with the control group, α-SMA and Collagen1 mRNA expression levels in the 5, 10 and 15 µmol/L NaAsO2 groups increased, and those in the 15 µmol/L NaAsO2 group were higher than those in the 10 and 5 µmol/L NaAsO2 groups; those in the 10 µmol/L Na‑ AsO2 group were higher than those in the 5 µmol/L NaAsO2 group (all P<0. 05). Compared with the control group, the mRNA expression levels of SLC7A11 and GPX4 in the 5, 10 and 15 µmol/L NaAsO2 groups decreased, and those in the 15 µmol/L NaAsO2 group were lower than those in the 10 and 5 µmol/L NaAsO2 groups; those in the 10 µmol/L NaAsO2 group were lower than those in the 5 µmol/L NaAsO2 group. The α-SMA mRNA was negatively correlated with GPX4 and SLC7A11 mRNA expression (r=-0. 72, -0. 92, respectively; both P<0. 01), and CollagenⅠ mRNA was negatively correlated with GPX4 and SLC7A11 mRNA expression (r=-0. 67, -0. 90, respectively; both P<0. 01). Conclusions With the increase of NaAsO2 concentration, the expression level of fibrosis gene increases gradually, which causes the activation of hepatic stellate cells. In this process, the down-regulated expression of SLC7A11 and GPX4 genes causes ROS accumulation and promotes ferroptosis in hepatic stellate cells. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of sodium arsenite (NaAsO2) on the cell viability and differentiation of mouse neural stem cells (NE-4C) induced by all-trans retinoic acid (ATRA). Methods NE-4C cells were induced by 0,0. 5,1. 0 and 5. 0 µmol/L ATRA, and the cell viability was detected by CCK-8. Neural stem cells (NSCs) specific markers ［Nestin and sex determining region Y-box 2(Sox2)］, neuron-specific markers ［neuron-specific enolase (NSE), RNA binding protein fox-1 homolog 1(Rbfox1), and βⅢ-Tubulin］, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) mRNA were detected by real-time quantitative fluorescent PCR (RT-PCR), and the appropriate con‐ centration of ATRA was screened. NE-4C cells were treated with ATRA at appropriate concentration to establish the neural stem cell differentiation models, which were then divided into the control group and experimental groups 1,2,3 and 4. Experimental groups 1,2,3 and 4 were treated with 0. 3,0. 6,0. 9 and 1. 2 µmol/L NaAsO2, respectively, while the control group was not treated with any drug. After 7 days of culture, CCK-8 method was used to detect cell viability, immunofluorescence staining was used to observe cell differentiation, and flow cytometry was used to detect Nestin, NSE,βⅢ -Tubulin and GFAP in cells. Results After ATRA treatment, the cell viability of NE-4C cells decreased, the mRNA ex‐ pression levels of Sox2 and Nestin decreased, and the mRNA expression levels of βⅢ-tutubulin, NSE and Rbfox1 in‐ creased, in a dose-dependent manner； the cell viability and the expression of differentiation-related genes were the highest in the treatment of 0. 5 µmol/L ATRA (P<0. 05). After the neural stem cell differentiation model was established, the cell viability of NE-4C cells and the expression levels of Nestin, NSE,βⅢ-Tubulin and GFAP in the control group and experimental groups 1,2,3 and 4 decreased successively (all P<0. 05). Conclusions NaAsO2 can decrease the cell viability of NE-4C cells induced by ATRA and inhibit the differentiation of NE-4C cells into neurons. The higher the concentration of NaAsO2, the stronger the inhibitory effect on the cell viability and differentiation of NE-4C cells. [ABSTRACT FROM AUTHOR]

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BackgroundLong-term exposure to arsenic can cause liver injury of varying degrees. Mitochondrial damage may be an early key event of arsenic-induced liver injury. Silent mating type information regulation 2 homolog 1 (SIRT1)/ recombinant peroxisome proliferators-activated receptor gamma coactivator 1 alpha (PGC-1α) is an important pathway regulating mitochondrial mass and function. However, whether arsenic-induced liver injury is related to mitochondrial dysfunction mediated by SIRT1/PGC-1α pathway remains unclear.ObjectiveTo investigate potential effects of sodium arsenite (NaAsO2) on mitochondrial function and expressions of SIRT1/PGC-1α pathway-related proteins in human normal liver cell.MethodsHuman normal liver cells (MIHA cells) were used as the research object. MIHA cells were treated with different concentrations of NaAsO2 (0, 5, 10 and 20 μmol·L−1) for 24 h, and the cells were collected for study. The ultrastructure of mitochondria was observed by transmission electron microscopy, adenosine triphosphate (ATP) concentration by fluorescence method, mitochondrial membrane potential (MMP) level by flow cytometry, and SIRT1, PGC-1α and their downstream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) protein expression levels by Western blotting. One-way analysis of variance and trend test were used for data statistical analysis.ResultsThe viability of MIHA cells decreased gradually with the increase of NaAsO2 concentration (F=6495.47, P

ObjectiveTo investigate the effect of subchronic arsenic exposure on hepatic glucose metabolism and its molecular mechanism in rats. MethodsTotally 40 Sprague-Dawley (SD) rats were randomly divided into a control group (with intragastric administration of distilled water) and three groups with intragastric administration of sodium arsenite (NaAsO2) at doses of 2, 4, 8 mg/kg once a week continuously for 12 weeks. The body weight of the rats was recorded weekly. By the end of the administrations, oral glucose tolerance test (OGTT) was performed and samples of hair and liver were collected for all the rats. The content of arsenic in hair, the content of glycogen in liver and the activities of hexokinase (HK) and pyruvate kinase (PK) were detected. Ppathological changes of liver were observed. The expressions of phosphoinosistide 3-kinase (PI3K), protein kinase B (AKT), glycogen synthase kinase-3 β (GSK3β), phosphorylated glycogen synthase kinase-3 β(P-GSK3β), glucose transporter 2 (GLUT2) and glucose transporter 4 (GLUT4) were determined with Western blot. ResultsThe body weight decreased at 8th and 12th week for the rats exposed to high and moderate NaAsO2. Significantly higher hair arsenic content (mg/kg) was measured in the rats exposed to high/moderate/low NaAsO2 compared with that of control rats (49.24 ± 8.02/21.06 ± 3.42/14.59 ± 2.00 vs. 0.43 ± 0.08; all P < 0.05). Abnormal glucose tolerance and decreased liver glycogen were also detected in the rats with NaAsO2 exposure. In comparison to control rats, the rats treated with high/moderate NaAsO2 had significantly decreased HK (1.94 ± 0.11/2.14 ± 0.03 vs. 2.84 ± 0.08) U/mg and PK (43.64 ± 1.05/44.26 ± 0.10 vs. 55.95 ± 0.96) U/mg (P < 0.05 for all). Pathological observation revealed mild hepatic steatosis and increased round vacuoles of hepatocytes in the rats with high and moderate NaAsO2 treatment. Contrasted to the protein expressions of PI3K (1.03 ± 0.02), AKT (1.36 ± 0.02), P-GSK3β/GSK3β (0.96 ± 0.04), GLUT2 (1.24 ± 0.12), and GLUT4 (1.80 ± 0.15) in the control rats, following significantly decreased protein expressions were identified in the rats exposed to various doses of NaAsO2: PI3K (0.76 ± 0.06), AKT (1.19 ± 0.04), P-GSK3β/GSK3β(0.76 ± 0.03), GLUT2 (0.82 ± 0.11), and GLUT4 (0.88 ± 0.14) in the rats treated with high dose; AKT (1.08 ± 0.10) and GLUT4 (1.38 ± 0.10) in the rats with moderate dose; and GLUT4 (1.10 ± 0.12) in the rats with low dose (all P < 0.05 ). ConclusionSubchronic arsenic exposure can induce hepatic glucose metabolism disorder in rats; the effect may be related to changes in hepatic glycogen, activities of glucose metabolic enzymes, and expressions of relevant proteins.

BackgroundThe key enzymes of serine synthesis pathway (SSP) play an important role in tumor growth, proliferation, and invasion, but their roles in arsenic carcinogenesis are unclear.ObjectiveTo observe the effects of NaAsO2 treatment on the expressions of key enzymes [such as phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)] of SSP and on the ability to proliferate and migrate in human immortalized skin keratinocytes (HaCaT) and NaAsO2-induced malignantly transformed HaCaT (T-HaCaT), and to explore the roles of SSP key enzymes in arsenic carcinogenesis.Methods(1) The T-HaCaT cells constructed earlier by our research team were divided into a passage control (0 μmol·L−1 NaAsO2) group, a T-HaCaT (0.5 μmol·L−1 NaAsO2) group, a NCT503 (PHGDH inhibitor, 25 μmol·L−1) group, and a NCT503 (25 μmol·L−1) + T-HaCaT (0.5 μmol·L−1 NaAsO2) group. Western blotting was used to detect the protein expression levels of SSP key enzymes in the passage control group and the T-HaCaT group. CCK8 assay and cell scratch test were used to detect the proliferation and migration rates of cells in each group respectively. (2) Well-grown logarithmic-phase HaCaT cells were treated with 0, 0.625, 1.25, and 2.5 μmol·L−1 NaAsO2 for 0, 24, 48, and 72 h to detect cell proliferation rate and protein expression levels of SSP key enzymes. In the subsequent experiment, HaCaT cells were pretreated with 25 μmol·L−1 NCT503 for 6 h, and then treated with 2.5 μmol·L−1 NaAsO2 for 72 h continuously. The experimental groups included a control (0 μmol·L−1 NaAsO2) group, an exposure (2.5 μmol·L−1 NaAsO2) group, a pretreatment (25 μmol·L−1 NCT503) group, and a pretreatment (25 μmol·L−1 NCT503) + exposure (2.5 μmol·L−1 NaAsO2) group, to detect the proliferation rate of cells in each group.ResultsThe protein expression level of PHGDH in the T-HaCaT group were 1.60 times higher than that in the passage control group (P

BackgroundIn addition to the typical signs of skin damage, long-term arsenic exposure is often accompanied by signs and symptoms of neurobehavioral abnormalities.ObjectiveTo investigate potential intervention effect of sciadopitysin on senescence of neurons induced by sodium arsenite in rats and possible underlying mediating effect of cell cycle-related transcription factor E2F1.MethodsSH-SY5Y cells were treated with 4 μmol·L−1 sodium arsenite for 24 h and intervened with 50 μg·mL−1 Ginkgo biloba extract (EGb761) or four major biflavonoids in Ginkgo biloba leaves (isoginkgetin, bilobetin, sciadopitysin, and ginkgetin) for 24 h respectively. Then, cell viability was measured by CCK-8 assay. Thirty-two 180-200 g SPF rats were randomly divided into a control group, an arsenic treatment group (10 mg·L−1), a Ginkgo biloba extract intervention group (10 mg·kg−1), and a sciadopitysin intervention group (10 mg·kg−1), 8 rats in each group, half male and half female. The rats were treated with sodium arsenite by free drinking water for 3 consecutive months, and the intervention treatment was conducted after 2 months of poisoning with drug intake by gavage for 1 month. HE staining was used to detect structural changes in the hippocampus, while Nissl's staining was used to detect changes in hippocampal morphology and neuron numbers. Moreover, senescence-associated β galactosidase (SA-β-gal) staining and Western blotting were used to detect senescence of hippocampal neurons and the expression level of E2F1, respectively.ResultsCompared to the arsenic treatment group, EGb761 and the four biflavonoids in Ginkgo biloba leaves effectively antagonized the inhibitory effect of sodium arsenite on cell viability (all Ps＜0.05), and sciadopitysin showed better restoration of cellular viability than Ginkgo biloba extract (P＜0.05). The results of HE staining and Nissl's staining showed that the hippocampal neurons in the arsenic treatment group were reduced in cell count and the synaptic structure was abnormal, with swelling, nuclear shrinkage, and vacuole, compared with the control group. The results of SA-β-gal staining showed that the number of senescent cells in the arsenic treatment group (15.75±3.01) was significantly increased compared with the control group (2.88±0.84) (P＜0.05); the numbers of senescent cells in the Ginkgo biloba extract group (9.38±1.92) and the sciadopitysin treatment group (7.75±2.38) were significantly decreased compared with the arsenic treatment group (all Ps＜0.05). The results of Western blotting showed that compared with the control group, the expression of E2F1 protein in hippocampus of the arsenic treatment group was significantly decreased (1.00±0.17 vs. 0.65±0.19, P

BackgroundArsenic is a well-known environmental toxicant. Hepatic fibrosis could occur dueto excessive or long-term exposure to arsenic, while associated molecular mechanisms remain undefined. Mitogen-inducible gene 6 (Mig-6) exhibits a protective effect on numerous diseases or cancers. However, the specific role of Mig-6 in the mechanisms of arsenite-induced hepatic fibrosis remains indistinct.ObjectiveTo investigate the specific role of Mig-6 in the activation of hepatic stellate cells (HSC) and the deposition of extracellular matrix (ECM) induced by sodium arsenite (NaAsO2).MethodsHuman hepatic stellate cells (Lx-2) were treated with 0, 1.875, 3.75, 7.5, and 15 μmol·L−1 of NaAsO2 for 24 h, or with 7.5 μmol·L−1 NaAsO2 for 0, 12, 24, 48, and 72 h. Additionally, Lx-2 cells were transfected by pcDNA3.1(+)/Mig-6, then treated with 7.5 μmol·L−1 NaAsO2 for 24 h; a blank control group, a pcDNA3.1(+)-control group, a pcDNA3.1(+)/Mig-6 group, and an arsenic (7.5 μmol·L−1 NaAsO2) group were also set up. After transfection, the cells and culture supernatants were collected, and the protein levels of Mig-6, α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) in Lx-2 cells were identified by Western blotting analysis; moreover, the secretion levels of main ECM components in supernatants such as hyaluronic acid (HA), laminin (LN), collagens IV (COL-IV), and procollagen-III (PIIINP) were tested by ELISA. ResultsThe Mig-6 expression decreased in the 3.75, 7.5, and 15 μmol·L−1 NaAsO2 groups (0.561±0.095, 0.695±0.048, and 0.401±0.030) compared to the control group (1.000±0.000) in Lx-2 cells (P

To investigate the effects of different doses of sodium arsenite exposure on human hepatocellular carcinoma cell line LM3, and analyze the underlying potential molecular mechanisms of sodium arsenite-mediated promotion of malignant migration of LM3 cells. Methods • The model of sodium arsenite exposure was established by continuously culturing LM3 cells in high DMEM glucose medium containing 0, 1 and 10 pmol/L sodium arsenite for 14 weeks. After exposure, the sodium arsenite-containing cell culture medium was discarded, and the cells were allowed to recover in sodium arsenite-free culture medium for one week. Cells from the same batch without sodium arsenite treatment were used as the control group. Subsequently, CCK-8 proliferation assay, Transwell migration assay, and DCFH-DA reactive oxygen species fluorescence probe experiments were performed to verify the effect of chronic sodium arsenite exposure on LM3 cell proliferation, migration, and oxidative stress. Realtime quantitative PCR and Western blotting were performed to elucidate the effect of chronic sodium arsenite exposure on the expression of mRNA and protein related to tumor metastasis in LM3 cells. Results • Compared with the control group, the sodium arsenite exposure groups (1 pmol/L and 10 pmol/L) had significantly higher reactive oxygen species (ROS) levels (both F>0.05), and sodium arsenite exposure promoted the malignant migration of LM3 cells in a dose-dependent manner (both P>0.05), but sodium arsenite had no significant effect on the proliferation of LM3 cells. Sodium arsenite exposure up-regulated the gene expression levels of vascular endothelial growth factor (VEGF), nicotinamide adenine dinucleotide phosphate oxidases 2 (NOX2), and mitogen-activated protein kinase 8 (MAPK8), and up-regulated the protein expression level of NOX2 and MAPK8. Conclusions • Sodium arsenite exposure can promote malignant migration and oxidative stress level of LM3 cells, and up-regulate the expression levels of VEGF, NOX2 and MAPK8 genes, suggesting that the mechanism of sodium arsenite promoting malignant migration of LM3 cells may be related to oxidative damage and up-regulation of these genes. [ABSTRACT FROM AUTHOR]

BACKGROUND: As the main target organ of arsenic toxicity, the liver has become the focus of research on the mechanism of arsenic toxicity. OBJECTIVE: To investigate the effects of sodium arsenite (NaAsO2) on oxidative stress and apoptosis of AML12 hepatocytes, as well as the influence on the expression of key molecules in the Hippo signaling pathway. METHODS: AML12 hepatocytes were cultured in medium containing 0, 10, 15, 20, 25, 30 μmol/L NaAsO2 for 24 hours. The cell morphology was observed by phase-contrast microscopy and cell viability was assessed by cell counting kit-8 assay. The content of intracellular reactive oxygen species was detected by fluorescent probe technique combined with flow cytometry. The activity of caspase-3 was measured by colorimetry. The apoptotic rate of hepatocytes was detected by annexin V/propidium iodide double staining combined with flow cytometry. The mRNA expression level of Yes-associated protein was detected by real-time fluorescence quantitative PCR. The protein expression level of phosphorylated mammalian sterile20-like kinases1/2, phosphorylated Yes-associated protein and Yes-associated protein were detected by western blot assay. RESULTS AND CONCLUSION: Compared with the control group, the viability of AML12 hepatocytes decreased gradually along with the increase of NaAsO2 concentration. With the increase of NaAsO2 concentration, the reactive oxygen species contents, the activity of caspase-3 and the apoptotic rate increased gradually in AML12 hepatocytes. There was no significant change in the mRNA expression of Yes-associated protein in NaAsO2 treated AML12 hepatocytes. NaAsO2 elevated the relative protein expression of phosphorylated mammalian sterile20-like kinases 1/2 and phosphorylated Yes-associated protein, while inhibited the relative protein expression of Yes-associated protein. Therefore, NaAsO2-induced oxidative stress and apoptosis injury in AML12 hepatocytes may be related to the activation of Hippo signaling pathway. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of sodium arsenite (NaAsO2) on activation of human hepatic stellate cells（LX-2 cells) and its mechanism. Methods LX-2 cells were cultured in vitro. The LX-2 cells in the logarithmic growth phase were cultured with NaAsO2 medium at the concentrations of 0，5，15，20，30，40，50 µmol/L. The cell viability was detected by MTT method and the experimental concentration of NaAsO2 was determined. LX-2 cells in the logarithmic growth phase were randomly divided into the control group, low-，medium- and high-concentration NaAsO2 groups， which were treated with 0，5，10，and 15 µmol/L NaAsO2 for 48 h, respectively. The autophagy of the cells was detected by transmission electron microscope, and the expression levels of the cell activated protein (ɑ-SMA) and autophagy protein（AKT，Hes1，and mTOR) were detected by Western blotting. Results With the increase of NaAsO2 concentration， the inhibition rate of cell proliferation increased gradually. Five，10，and 15 µmol/L were selected as the experimental concentrations of NaAsO2 according to the half-maximal inhibitory concentration and the cell viability. Autophagosome and autophagolysosome were observed in the control group. In the low-，medium- and high-concentration NaAsO2 groups， the cell gap widened graduallym and "False Dee gap" was observed. The number of autophagosomes increased, and the distribution of lipid droplets was more dense. Some mitochondria were swollen and vacuolated, and a large number of mitochondrial cristae were broken, and the above changes were the most obvious in the high-concentration NaAsO2 group. Compared with the control group, the relative expression of ɑ-SMA protein gradually increased, the protein expression of AKT and mTOR gradually decreased, and while the protein expression of Hes1 gradually increased in the low-, medium and high-concentration NaAsO2 groups（all P<0. 05）. Conclusion NaAsO2 may induce the activation of LX-2 cells by promoting autophagy. [ABSTRACT FROM AUTHOR]

Objective: To explore the role of nuclear transcription factor E2-related factor 2(NRF2)-mediated reductive stress in arsenite induced malignant transformation in human keratinocytes., Methods: HaCaT cells and fluorescent labeled mitochondrial glutathione HaCaT cells(Mito-Grx1-roGFP2 HaCaT) were cultured to 35 passages in medium containing 0.0 and 1.0 μmol/L NaAsO&#95;2 to establish a model of malignant transformation of cells. Cellular and mitochondrial reduced glutathione/oxidized glutathione(GSH/GSSG) and reduced coenzyme II/oxidized coenzyme II(NADPH/NADP~+) ratios were measured in HaCaT cells. Cell doubling time, cell migration ability, soft agar clone formation ability and GSH/GSSG at different times in the 0 passage, the early stage(1st, 7th and 14th passages) and later stage(21st, 28th and 35th passages) were measured in Mito-Grx1-roGFP2 HaCaT cells. NaAsO&#95;2 induced malignant transformation cells were transfected with NRF2 siRNA, and detected the expression level of NRF2 and the redox-related indexes and malignant transformation indexes., Results: Compared with the control group, the GSH/GSSG ratio in 1.0 μmol/L NaAsO&#95;2 treated HaCaT cells significantly decreased in the 1st and 7th generations, but significantly increased after the 21st generation, and the NADPH/NADP~+ ratio significantly increased in the 1st, 14th, 21st, 28th and 35th generations; The levels of GSH/GSSG in mitochondria significantly increased from 1st to 35th generation, and the levels of NADPH/NADP~+ in mitochondria significantly increased at 1st, 7th, 21st, 28th and 35th generation. After continuous treatment of Mito-Grx1-roGFP2 HaCaT cells with 0.0 or 1.0 μmol/L NaAsO&#95;2 to 35 passages, the doubling time of cells treated with 1.0 μmol/L NaAsO&#95;2 was significantly shortened, the cell migration rate was increased greatly, and more clones with larger volumes than the control cells formed. The GSH/GSSG ratio in mitochondria of Mito-Grx1-roGFP2 HaCaT cells showed a significant decrease in the 1st generation and increased from the 7th generation onwards(all P&lt;0.05). After transfection of NaAsO&#95;2 treated cells with NRF2 siRNA, the levels of hydrogen peroxide and superoxide increased compared with the siRNA controls. The levels of cell and mitochondrial NADPH/NADP~+ and GSH/GSSG decreased and the level of mitochondrial GSH/GSSG in Mito-Grx1-roGFP2 HaCaT cells decreased. Cell doubling time increased, cell migration rate and soft agar clone formation ability decreased(all P&lt;0.05). The malignant phenotype was reversed., Conclusion: In the early stage(1st, 7th and 14th passages) of NaAsO&#95;2 treated HaCaT cells, oxidative stress occurred with continuous high NRF2 expression. Later(21st, 28th and 35th passages), NRF2 induced reductive stress, leading to malignant transformation.

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Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik . Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly ( P <0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P -Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P <0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences ( P <0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference ( P =0.024) , but there was no significant difference in the expression level of Bik mRNA ( P =0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups ( P =0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups ( P =0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups ( P =0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.

Objective: To explore the effects of sodium arsenite (NaAsO(2)) exposure on the activation and extracellular matrix secretion of human hepatic stellate cells, and to provide a theoretical basis for the mechanism study of arsenic induced hepatic fibrosis. Methods: Different doses of NaAsO(2) (0.0, 0.1, 1.0, 10.0, 50.0, 100.0 μmol/L) were exposed to human hepatic stellate cell line (Lx-2) for 24, 48 and 72 huors. CCK-8 assay was used to measure cell viability and IC(50) of NaAsO(2) on Lx-2 was then calculated; According to IC(50) results, 0.000, 1.875, 3.750, 7.500, and 15.000 μmol/L of NaAsO(2) were exposed to Lx-2 cells for 24 hours, besides, 7.500 μmol/L of NaAsO(2) was exposed to Lx-2 cells for 0, 12, 24, 48, and 72 hours, then collected cells and culture supernatant; HSC activation-related protein, including α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) expression levels were detected by Western blot analysis, the main extracellular matrix including laminin (LN) , hyaluronic acid (HA), collagen Ⅳ (COL-Ⅳ) and procollagen Ⅲ(P Ⅲ NP) secretion level was detected by Elisa assay. Results: CCK-8 assay showed that the cell viability of Lx-2 cells were increased obviously at low doses (≤1.0 μmol/L) of arsenic exposure, especially at 48 and 72 h. In contrast, with the increasing doses of arsenic exposure, the survival rate of Lx-2 cell was decreased gradually, and the survival rate of the high-dose (50, 100 μmol/L) arsenic exposure group at 24, 48 and 72 h were significantly lower than 0.0 μmol/L group, P< 0.05. The IC(50) of NaAsO(2) on Lx-2 cells at 24, 48, 72 h were calculated as 72.75, 48.19 and 29.95 μmol/L, respectively; The expression levels of HSC activation-related protein showed that, after treated with 1.875, 3.750, 7.500, 15.000 μmol/L NaAsO(2) for 24 h, α-SMA and TGF-β1 protein level were higher than 0.000 μmol/L group. The increased expression of α-SMA and TGF-β1 protein were most significant in 7.500 μmol/L NaAsO(2) group ( P< 0.05). In addition, the expression levels of α-SMA and TGF-β1 also showed a time-dependent increasing in Lx-2 cells after treated with 7.500 μmol/L NaAsO(2) for 0, 12, 24, 48 and 72 h; Elisa assay showed that after treated with 1.875, 3.750, 7.500, 15.000 μmol/L NaAsO(2) for 24 h, the secretion levels of HA, LN, COL-Ⅳ and PⅢNP were obvious higher than 0.000 μmol/L group ( P< 0.05). Moreover, the secretion levels of HA, LN, COL-Ⅳ and P Ⅲ NP also showed a time-dependent increased manner in Lx-2 cells after exposed to 7.500 μmol/L NaAsO(2) for 0, 12, 24, 48 and 72 h ( P< 0.05). Conclusion: NaAsO(2) exposure to Lx-2 cells can upregulate the expression level of HSC activation-related proteins, induce its further activation, then increase ECM secretion level.

Objective: To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study. Methods: Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD(50)) was calculated in linear regression. Results: Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD(50) of lead acetate was 2 025.0 μmol/L, the LD(50) of cadmium chloride was 36.6 μmol/L, and the LD(50) of sodium arsenite was 33.2 μmol/L. Conclusion: The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.

Objective: To determine the effects of arsenic and estrogen receptor antagonist (ICI182, 780) on the expression of estrogen receptor beta (ERβ) in alveolar Ⅱ epithelial cells (AECⅡ) of female and male mice., Methods: Nineteen or twenty day fetus mice were obtained through caesarean section of ICR mice. Purified AECⅡ cells were separated from the female and male fetus,respectively,and confirmed using immunofluorescence staining. The cells were exposed to sodium arsenite (NaAsO 2 ) at a low,medium,or high dosage determined by MTT and cultured for 24 h. The NaAsO 2 (5 μmol/L) exposed cells were compared with those treated (for 24 h) with dimethyl sulfoxide (DMSO) or ICI182, 780 (1×10 -4 mol/L). Apoptosis rates of the cells were measured by flow cytometry. Real-time fluorescence quantitative PCR method and Western blot technique were used to detect the expression of ERβ mRNA and protein in AECⅡ., Results: Purity of AECⅡ cells reached (87.0±2.5)%. NaAsO 2 exposure was set at a concentration of 0.5 (low),1.25 (medium),and 5 (high) μmol/L. The cells exposed to medium and high dosage of NaAsO 2 had higher apoptosis rates than the blank controls ( P <0.05),without sex differences. Female cells exposed to medium and high dosage of NaAsO 2 had higher levels of expressions of ERβ mRNA and protein than the blank controls ( P <0.05) and male cells exposed to the same dosage of NaAsO 2 ( P <0.05). No significant differences were found in the expressions of ERβ mRNA and protein between the exposed male cells and the blank controls. ICI182, 780 lowered the expression levels of ERβ mRNA and protein in the female exposed cells ( P <0.01)., Conclusion: Arsenic exposure increases expressions of AECⅡ's ERβ,more so in female cells than in male cells. This can be blocked by estrogen receptor antagonists., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Objective: To investigate the effect of chronic exposure to sodium arsenite at a dose of 1. 0 μmol / L on proliferation of human bronchial epithelial cells( HBE) and human keratinocytes( HaCaT) and discuss the mechanism of arsenic carcinogenesis., Methods: Malignant transformation model of HBE and HaCaT cells cultured in vitro were used in this study. MTT assay was used to detect the capacity of proliferation. Flow cytometry was used to detect cell cycle. The expression of cell cycle related protein like cyclin E, cyclin D1 and cyclin A protein were inspected by Western blot., Results: The treated cells, including passage 36 and 43 of HBE cells and passage 28 and 35 of HaCaT cells grow faster than the control group( P < 0. 01 and P < 0. 05). The treated cells in the G1 phase were decreased( P < 0. 05), however cells in the S phase were increased( P <0. 05). In addition, the expression of cyclin E displayed a trend of up-regulation( P <0. 05), and it was maintained at a high level in advanced period., Conclusion: By increasing the expression of cyclin E in HBE and HaCaT cells, low dose of sodium arsenite made cells escaping from the G1 phase to S phase, accelerating cell cycle progression and proliferation, a way that may lead to malignant transformation.