ABSTRACT
Background Long-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. Objective To investigate potential effects of sodium arsenite (NaAsO2) on mitochondrial function and expressions of SIRT1/PGC-1α pathway-related proteins in human normal liver cell. Methods Human 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. Results The viability of MIHA cells decreased gradually with the increase of NaAsO2 concentration (F=6495.47, P<0.001). The transmission electron microscope observation showed that the size of mitochondria in the 10 μmol·L−1 NaAsO2 treatment group was different, and the mitochondria were swollen or elongated in a rod-like shape. The mitochondria in the 20 μmol·L−1 NaAsO2 treatment group swelled like air spheres or vacuoles. The ATP concentration and MMP level of MIHA cells gradually decreased with the increase of NaAsO2 concentration (Ftrend of ATP=172.28, Ftrend of MMP=59.91, both Ps<0.001). Compared with the control group, the protein expression levels of SIRT1, PGC-1α, NRF1, and TFAM were not significantly changed in the 5 μmol·L−1 NaAsO2 treatment group, while the protein expression levels of SIRT1, PGC-1α, and TFAM were decreased in the 10 μmol·L−1 NaAsO2 treatment group, and the protein expression levels of SIRT1, PGC-1α, and NRF1 were decreased in the 20 μmol·L−1 NaAsO2 treatment group. The results of trend test showed that the protein expression levels of SIRT1, PGC-1α, NRF1, and TFAM decreased gradually with the increase of NaAsO2 concentration (Ftrend of SIRT1=47.07, P<0.001; Ftrend of PGC-1α=15.17, P<0.01; Ftrend of NRF1=13.54, P<0.01; F trend of TFAM=4.20, P<0.05). Conclusion The down-regulation of SIRT1/PGC-1α and its downstream NRF1 and TFAM may be involved in NaAsO2-induced mitochondrial dysfunction in liver cells.
ABSTRACT
Background In addition to the typical signs of skin damage, long-term arsenic exposure is often accompanied by signs and symptoms of neurobehavioral abnormalities. Objective To 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. Methods SH-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. Results Compared 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<0.05); compared with the arsenic treatment group, the protein expression level of E2F1 in hippocampus of the sciadopitysin treatment group (0.89±0.18) was significantly recovered (P<0.05); compared with Ginkgo biloba extract (0.68±0.19), sciadopitysin had a better recovery effect on E2F1 expression level (0.89±0.18) (P＜0.05). The results of correlation analysis showed that the E2F1 protein expression level was negatively correlated with the positive rate of SA-β-gal staining in hippocampal neurons (r=−0.518, P＜0.05). Conclusion Sciadopitysin is an effective component of Ginkgo biloba extract. It can effectively inhibit the senescence of hippocampal neurons induced by sodium arsenite, and E2F1 may play an important mediating role.
ABSTRACT
Background The 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. Objective To 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. Results The protein expression level of PHGDH in the T-HaCaT group were 1.60 times higher than that in the passage control group (P<0.05), and its proliferation rate (177.51%±14.69%) and migration rate (53.85%±0.94%) were also higher than the passage control group’s (100.00%±0.00% and 24.30%±2.26%) (both Ps<0.05), respectively. After the NCT503 intervention, the proliferation rate (144.97%±8.08%) and migration rate (35.80%±0.99%) of cells in the NCT503 + T-HaCaT group were lower than those in the T-HaCaT group (both P<0.05). The proliferation rate of HaCaT cells after NaAsO2 exposure for 72 h increased with the increase of exposure concentration (r=0.862, P<0.05), and consistently, the protein levels of SSP key enzymes in HaCaT cells in each exposure group were higher than those in the control group (all P<0.05). The proliferation rate of HaCaT cells treated with 2.5 μmol·L−1 NaAsO2 increased with the extension of exposure time (r=0.775, P<0.05), which was consistent with the changes of PHGDH levels in cells. After the NCT503 intervention, the proliferation rate of the pretreatment + exposure group was significantly lower than that of the exposure group (P<0.05). Conclusion The key enzymes of SSP may play an important role in the proliferation of T-HaCaT cells induced by NaAsO2.
ABSTRACT
Background Arsenic 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. Objective To 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). Methods Human 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. Results The 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<0.05). After administration with 7.5 μmol·L−1 of NaAsO2 for 24, 48, and 72 h, the Mig-6 expression (0.856±0.036, 0.515±0.077, 0.491±0.060) decreased compared with the 0 h group (1.000±0.000) (P<0.05). After over-expression of Mig-6, the results of Lx-2 activation related protein levels showed that compared to the control group, the α-SMA and TGF-β1 expression were up-regulated in the arsenic group (P<0.05); meanwhile, the α-SMA and TGF-β1 in the Mig-6 over-expression combined arsenic exposure group reduced compared to the arsenic (7.5 μmol·L−1) group (P<0.05). The results of ELISA showed that compared with the control group, the HA, LN, PIIINP, COL-IV in the arsenic group were up-regulated (P<0.05); while compared to the arsenic group, the HA, LN, PIIINP, and COL-IV in the Mig-6 over-expression combined with arsenic exposure group were decreased (P<0.05). Conclusion Arsenic down-regulates Mig-6 expression in HSC, and over-expression of Mig-6 can reverse the activation of HSC and ECM deposition induced by arsenic exposure. It suggests that Mig-6 plays a protective role in arsenic-induced HSC activation and ECM deposition.
ABSTRACT
Objective@#To examine the effect of chronic exposure to sodium arsenite on liver damages in rats. @*Methods@#Fifty-six healthy adult SD rats (28 males and 28 females) were randomly divided into 4 groups. Rats in the low-, medium- and high-dose groups were given sodium arsenite solutions at doses of 2, 10 and 50 mg/L for successive 24 weeks, while animals in the control group were given deionized water. The rat body and liver weights were measured and the liver coefficient was estimated. The urine arsenic level was detected using atomic fluorescence spectrometry, and hepatic tissue sections were stained with uranium acetate and lead citrate for morphological observations under an electron microscope. @*Results@#The body weights of both male and female rats appeared a tendency towards a rise with the duration of exposure to sodium arsenite (male rat: Wald χ2=3 610.621, P<0.001; female rat: Wald χ2=2 186.217, P<0.001, and there were no significant differences in the rat body weight 24 weeks post-exposure to sodium arsenite in each group, while there was an interaction between time and group (male rat: Wald χ2=15.874, P=0.001; Wald χ2=9.460, P=0.024). There were significant differences in the rat liver weight and liver coefficient in each group (male rat: F=18.964 and 29.968, both P<0.001; female rat: F=11.919 and 15.070, both P<0.001), with the lowest liver weight (10.17±1.15) g and liver coefficient (1.99±0.21)% measured in male rats in the high-dose group, and the highest liver weight (12.91±1.29) g and liver coefficient (4.10±0.56)% in female rats in the high-dose group. The median urine arsenic levels (interquartile range) were 25.60 (30.27), 146.56 (101.06), 1 034.68 (600.06) and 3 796.98 (19 966.89) μg/L in rats in the control, low-dose, medium-dose and high-dose groups, respectively (χ2=50.211, P<0.001), and the urine arsenic level was significantly higher in the medium- and high-dose groups than in the control group (both P<0.001). Hepatic edema was seen in rats in the low- and medium-dose groups, and hepatic edema, focal hepatic cell necrosis, hyperplasia of bile capillaries and peri-bile capillary endolysis were observed in rats in the high-dose group.@*Conclusions@#Chronic exposure to arsenic may cause morphological alterations of rat hepatic tissues, and the rat hepatic damage aggravates with the dose of exposure to arsenic.
ABSTRACT
Objective:To observe the effect of dictyophora polysaccharide (DIP) on PINK1/Parkin pathway mediated mitophagy induced by sodium arsenite (NaAsO 2) in human hepatocytes (L-02 cells). Methods:The L-02 cells in logarithmic growth phase and in good condition were divided into control group, NaAsO 2 group (10 μmol/L), DIP group (80 μg/ml), DIP + NaAsO 2 group (80 μg/ml DIP + 10 μmol/L NaAsO 2) , N-acetylcysteine (NAC) group (5 mmol/L), and NAC + NaAsO 2 group (5 mmol/L NAC + 10 μmol/L NaAsO 2). Western blotting was used to detect the expression levels of mitophagy related proteins p62, microtubule-associated protein 1 light chain 3 (LC3)Ⅱ/LC3Ⅰ, PINK1, and Parkin. The mitochondrial stucture and autophagosomes were observed by transmission electron microscope, the fluorescent probe method was used to detect the expression level of intracellular reactive oxygen species (ROS). Results:Compared with the control group, the protein expressions of p62, LC3 Ⅱ/LC3 Ⅰ, PINK1, and Parkin in NaAsO 2 group were higher ( P < 0.05); compared with the NaAsO 2 group, the protein expressions of p62, LC3 Ⅱ/LC3 Ⅰ, PINK1 and Parkin were lower in DIP, DIP + NaAsO 2, NAC, and NAC + NaAsO 2 groups ( P <0.05). According to the transmission electron microscope, compared with the control group, the mitochondria of L-02 cells in NaAsO 2 group were significantly damaged and the number of autophagosomes increased. Compared with NaAsO 2 group, the degree of mitochondrial swelling, vacuolar degeneration and the number of autophagosomes decreased in DIP + NaAsO 2 group. Compared with the control group (33 110.00 ± 2 191.28), the intracellular ROS level in NaAsO 2 group was higher (48 000.00 ± 2 395.31, P < 0.05); the level of intracellular ROS in DIP + NaAsO 2 group (38 670.00 ± 2 620.56) was significantly lower than that in NaAsO 2 group( P < 0.05), and there was no significant change compared with the control group ( P > 0.05). Conclusions:NaAsO 2 can induce PINK1/Parkin mediated mitophagy in L-02 cells. DIP can alleviate NaAsO 2 induced mitophagy. DIP may affect PINK1/Parkin mediated mitophagy induced by NaAsO 2 through the regulation of ROS.
ABSTRACT
OBJECTIVE：To study the i mprovement effects of α-lipoic acid on glucose metabolism disorder of insulin resistant HepG2 cells. METHODS ：The effects of 25-1 000 µmol/L α-lipoic acid on survival rate of human hepatoma cell HepG2 were determined by MTT assay so as to determine the concentration of α-lipoic acid. Negative control group ，insulin resistance group （1× 10－7 mol/L insulin ），combination resistance group （30 µmol/L sodium arsenite+ 1×10－8 mol/L insulin ），α-lipoic acid low- concentration，medium-concentration and high-concentration groups were set up. HepG 2 cells were treated with α-lipoic acid for 12 h and then cultured with corresponding concentration of sodium arsenite or/and insulin for 24 h. The glucose oxidase method was used to detect the glucose consumption ，colorimetric method was used to detect hexokinase activity and pyruvate kinase activity ， and anthrone method was used to detect glycogen content. Western blot assay was used to detect the protein expression of GLUT 4， p-GSK3β and GSK3β as well as the ratio of p-Akt/Akt and p-GSK3β/GSK3β. RESULTS：25，50，100 µmol/L α-lipoic acid had no significant effect on the survival rates of HepG 2 cells（P＞0.05），and survival rates of H epG2 cells were higher than 96％，so they were used as the low ，medium and high concentration for follow-up study. Compared with negative control group ，glucose consumption，the activities of hexokinase and pyruvate kinase ，glycogen content ，protein expression of GLUT 4 and p-GSK 3β，the ratio of p-Akt/Akt and p-GSK 3β/GSK3β were decreased significantly in insulin resistance group and combined resistance group， while the protein expression of GSK 3β was increased significantly（P＜0.05）. Compared with combination resistance group ，the glucose consumption （except for α-lipoic acid low- concentration group ），the activities of h exokinase（except for α-lipoic acid low-concentration and medium-concentration groups ） andpyruvate kinase （except for α-lipoic acid low-concentration com and medium-concentration groups ）， glycogen contents ， protein expression of GLUT 4 （except for α-lipoic acid mail：bliang163@163.com low-concentration group ）and p-GSK3β，the ratio of p-Akt/ Akt（except for α-lipoic acid low-concentration and medium-concentration groups ）and p-GSK 3β/GSK3β（except for α-lipoic acid low-concentration groups ）were increased significantly in α-lipoic acid groups ，while protein expression of GSK 3β（except for α-lipoic acid low-concentration and medium-concentration groups ） was decreased significantly （P＜0.05）；glycogen content ， protein expression of GLUT 4 and the ratio of p-GSK 3β/GSK3β in α-lipoic acid high-concentration group as well as the protein expression of p-GSK 3β in α-lipoic acid medium-concentration and high-concentration groups were improved significantly （P＜0.05）. CONCLUSIONS：α-lipoic acid can improve the disorder of glucose metabolism in insulin resistant HepG 2 cells，the mechanism of which may be associated with the increase of glucose consumption ，the activities of glucose metabolism related enzymes and glycogen content ，and expression up-regulation of the phosphorylation levels of Akt and GSK 3β protein，the expression of GLUT 4 and p-GSK 3β proteins，down-regulation of the expression of GSK 3β protein.
ABSTRACT
HIGHLIGHTS Sodium arsenite can cause neoplastic transformation in cells. Curcumin reduced cell viability and increased LDH activity in transformed Balb/c 3T3 cells. Curcumin caused DNA damage in transformed Balb/c 3T3 cells. Curcumin may play a protective role in sodium arsenite-induced toxicity.
Abstract Arsenic is a toxic substance that spreads widely around the environment and accumulates as metalloid in the earth's crust. Arsenic and its derivatives are found in drinking water, nutrients, soil, and air. Exposure to arsenic is associated with lung, blood, skin cancer and various lesions. Curcumin is a polyphenolic compound derived from Curcuma longa (turmeric) rhizome and is one of the main curcuminoids. Curcumin is known to be antioxidant, antibacterial, anti-inflammatory, analgesic effects. This study aimed to investigate the potential of sodium arsenite to transform embryonic fibroblast cells and to evaluate the cytotoxic and genotoxic effects of curcumin in neoplastic transformed cells. Neoplastic cells transformation was induced by sodium arsenite in Balb/c 3T3 cells at the end of 32 days. After transformation assay, the transformed cells were treated with various concentration of curcumin to evaluate cell viability, lactate dehydrogenase activity and DNA damage for 24h. The results revealed that curcumin decreased cell viability and increased the activity of lactate dehydrogenase enzyme in neoplastic transformed Balb/c 3T3 cells. In conclusion, the results demonstrated that curcumin has an anticancer effect on neoplastic transformed Balb/c 3T3 cells by causing DNA damage.
Subject(s)
Animals , Mice , Arsenic/toxicity , DNA Damage , Cell Transformation, Neoplastic , Curcumin/pharmacology , Fibroblasts/drug effects , BALB 3T3 Cells , Fibroblasts/pathologyABSTRACT
Aim To investigate the effects of sodium arsenite (NaAsO2) on the cell cycle and growth, and the intervention of MPST over-expression in the neural cells. Methods NaAsO2 was used to treat SH-SY5Y neuroblastoma cells for 48 h from the blank control (BC), empty vector control ( transfected with empty vector, NC ) and over-expression group ( lentiviral transfection with MPST,OP). The methods of CCK-8, crystal violet staining,flow cytometry and Western blot were used to examine cell viability,adherent rate,cell cycle and protein expression of p53,CDC25A,CyclinA and CDK2. Results The cell viability and adherent rate significantly decreased after treatment with NaAsO2 for 48 h,which was reversed in OP group (P <0. 01). Meanwhile, NaAsO2 also significantly increased the proportion of S phase cells and p53 protein expression, and down-regulated the protein levels of CDC25A,Cyc-lin A and CDK2 in BC and NC groups ( P < 0. 01), whereas the above changes of protein levels were significantly antagonized in OP group compared with NC group (P < 0. 05, P < 0. 01). Conclusions NaAsO2 inhibits the cell growth by inducing S-phase arrest and over-expression of MPST could reverse the noxious effects caused by NaAsO2 in SH-SY5Y cells.
ABSTRACT
Objective@#To explore the effects of sodium arsenite (NaAsO2) 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 NaAsO2 (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 IC50 of NaAsO2 on Lx-2 was then calculated; According to IC50 results, 0.000, 1.875, 3.750, 7.500, and 15.000 μmol/L of NaAsO2 were exposed to Lx-2 cells for 24 hours, besides, 7.500 μmol/L of NaAsO2 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 IC50 of NaAsO2 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 NaAsO2 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 NaAsO2 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 NaAsO2 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 NaAsO2 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 NaAsO2 for 0, 12, 24, 48 and 72 h (P<0.05).@*Conclusion@#NaAsO2 exposure to Lx-2 cells can upregulate the expression level of HSC activation-related proteins, induce its further activation, then increase ECM secretion level.
ABSTRACT
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 (LD50) 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 LD50 of lead acetate was 2 025.0 μmol/L, the LD50 of cadmium chloride was 36.6 μmol/L, and the LD50 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.
ABSTRACT
Objective: To evaluate the ameliorating effects of Raphanus sativus leaves (RSL) against sodium arsenite(Sa)-induced adverse effects through mice experiments. Methods: Swiss albino mice were divided into four equal groups: control, Sa, RSL, RSL + Sa. Sa (10 mg/kg body weight/day), and powder form of RSL (50 mg/kg body weight/day) were provided as food supplement orallty. Blood indices were measured using commercially available kits through colorimetric methods. Results: It was observed that lactate dehydrogenase, alkaline phosphatase, alanine aminotransferase,and aspartate aminotransferase activities were significantly(P<0.05) higher in Sa-treated mice than those in the control group.RSL significantly reduced Sa-induced elevation of the activities of these enzymes in serum significantly (P < 0.05). Serum butyrylcholinesterase activity and high density lipoproteins cholesterol levels in Sa-treated mice were significantly (P < 0.05) lower than the control group, and the food supplementation of RSL could significantly(P<0.05)prevent the reduction of Sa-mediated serum butyryl cholinesterase activity and high density lipoproteins cholesterol levels.RSL could also reduce the Sa-induced elevation of serum urea level significantly(P<0.05). Conclusions: Results of this study suggest the protective or ameliorating effects of RSL on Sa-induced perturbation of blood indices are related to the hepatic,cardiovascular and kidney dysfunction.Therefore,RSL may be useful to reduce arsenic toxicity in human in the future.
ABSTRACT
Objective To investigate the effect of sodium arsenite by Wnt signaling pathway on proliferation and apoptosis of oral squamous cell carcinoma.Methods Cell proliferation was detected after 1.25,2.5,5,10,20μmol/L sodium arsenite treatment human oral squamous cell carcinoma cell line Tca8113 for 24,48,72 hours by CCK8 experiment.0 and 14μmol/L sodium arsenite was used to treatment Tca8113 cells with 48h,cell apoptosis were detected by flow cytometry,Cleaved Caspase3,β-catenin,Cyclin D1 protein expression were detected by Western blot.Tca8113 cells were divided into control group,sodium arsenite group,activating agent+sodium arsenite group,all treated for 48hour,cell proliferation,apoptosis and Cleaved Caspase3,β-catenin,Cyclin D1 protein expression were detected by CCK8 assay,flow cytometry and Western blot.Results Tca8113 cell proliferation was inhibited significantly with the increase of treatment time and sodium arsenite concentration,and has a time and concentration dependent manner(P<0.05 or P<0.01).10μmol/L sodium arsenite as a follow-up study according to the IC50.Cell inhibition rate,apoptosis rate and Cleaved Caspase3 protein expression in 10μmol/L group were significantly higher than that of 0 mol/L group,the expression of β-catenin,Cyclin D1 protein was significantly lower than that of 0 mol/L group(P<0.01).Apoptosis rate,cell inhibition rate and Cleaved Caspase 3 protein expression in sodium arsenite group and activating agent+sodium arsenite group were significantly higher than control group,the expression of β-catenin and Cyclin D1 protein were significantly lower than control group(P<0.01).Apoptosis rate,cell inhibition rate and Cleaved Caspase 3 protein expression in activating agent + sodium arsenite group were significantly lower than that of sodium arsenite group,the expression of β-catenin and Cyclin D1 protein were significantly higher than that of sodium arsenite group(P<0.01).Conclusion Sodium arsenite can inhibit the proliferation of oral squamous cell carcinoma and promote apoptosis,and the mechanism was related to regulation of Wnt signaling pathway.
ABSTRACT
Objective To observe the effects of different levels of sodium arsenite ( NaAsO2) on mRNA expression of pigment epithelium-derived factor (PEDF) and apoptosis-related factors in PC12 cells ( rat neuron properties pheochromocytoma). Methods PC12 cells were treated with different levels of NaAsO2 [0 (control group), 2, 5, 10 μmol/L] for 24 hours. The mRNA expression of PEDF and apoptosis-related factors (Bax, Bcl-2) were detected by real-time quantitative PCR. Results There were significant differences in the mRNA expressions of PEDF between the 4 groups (F=102.28, P 0.05); there were significant differences in the mRNA expressions of Bcl-2 between the 4 groups (F=19.87, P<0.05), the mRNA expressions of Bcl-2 in the group of 2, 5, 10 μmol/L (0.65 ± 0.03, 0.49 ± 0.04, 0.57 ± 0.09) were lower than that of control group (0.95 ± 0.11, all P<0.05);there were significant differences in the mRNA expressions of Bax/Bcl-2 between the 4 groups (F=8.352, P<0.05), the mRNA expressions of in the group of 5, 10μmol/L (1.80 ± 0.72, 1.82 ± 0.36) were higher than that of control group (1.02 ± 0.24, all P<0.05). Conclusion NaAsO2 may increase the expression of apoptosis-related factorsBax/Bcl-2 mRNA by decreasing the expression of PEDF mRNA in PC12 cells, leading to apoptosis in PC12 cells.
ABSTRACT
Objectives To investigate the effects of sodium arsenite (NaAsO2) on the expression ot microRNA-191 (miR-191) and tissue inhibitor of metalloproteinase 3 (TIMP-3) in human normal hepatic cells (L-02 cells).Methods L-02 cells were exposed to different doses of NaAsO2 [0 (control group),5,25,50 and 75 μmol/L]for 24 h,or treated with 5 and 25 μmol/L NaAs02 for 0 (control group),12,24 and 48 h.The miR-191 inhibitor was used to suppress the expression of miR-191.qRT-PCR was performed to detect the expression level of miR-191 and TIMP-3 mRNA,and the protein level of TIMP-3 was analyzed by Western blotting.Results Dose-effect study:There were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the 5 groups (F =85.674,20.952,123.393,all P ＜ 0.05).The expressions of miR-191 in all groups (1.702 ± 0.124,2.077 ±0.234,2.145 ± 0.105,2.003 ± 0.077) were higher than that of control group (0.990 ± 0.035,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 25,50,75 μmol/L groups (0.848 ± 0.067,0.804 ± 0.081,0.813 ± 0.076) were all lower than that of control group (0.996 ± 0.007,all P ＜ 0.05),but there was no significant difference in the mRNA expression of TIMP-3 between the 5 μmol/L group and control group (0.939 ± 0.133 vs 0.996 ± 0.007,P＞ 0.05),and the protein expressions of TIMP-3 in all groups (0.846 ± 0.093,0.611 ± 0.123,0.554 ± 0.098,0.529 ± 0.067) were lower than that of control group (1.006 ± 0.003,all P ＜ 0.05).Time-effect study:there were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the exposure groups of 5 and 25 μmol/L (For 5 μmol/L:F =86.355,16.404,22.898,all P ＜ 0.05;For 25 μmol/L:F =104.321,20.123,52.321,all P ＜ 0.05).The expressions of miR-191 in all exposure groups of 5 and 25 μmol/L (1.392 ± 0.152,1.691 ± 0.167,2.018 ± 0.130 and 1.456 ± 0.167,1.946 ± 0.178,2.259 ± 0.256) were higher than those of control groups (1.001 ± 0.014,1.008 ±0.027,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 48 h exposure group of 5 μmol/L and all exposure groups of 25 μmol/L (0.824 ± 0.093 and 0.897 ± 0.033,0.815 ± 0.089,0.709 ± 0.103) were lower than those of control groups (1.004 ± 0.018,0.997 ± 0.057,all P ＜ 0.05),but there were no significant differences in the mRNA expressions of TIMP-3 between the 12,24 h exposure groups of 5 μmol/L and control group (0.952 ± 0.072,0.929 ± 0.121 vs1.004 ± 0.018,all P ＞ 0.05);the protein expressions of TIMP-3 in all exposure groups of 5 and 25 μmol/L (0.857 ±0.068,0.832 ± 0.106,0.691 ± 0.112 and 0.785 ± 0.097,0.620 ± 0.066,0.453 ± 0.075) were lower than those of control groups (1.006 ± 0.045,1.004 ± 0.078,all P ＜ 0.05).The treatment of miR-191 inhibitor:there were significant differences in the expressions of miR-191 and TIMP-3 protein between different groups (F =104.306,67.015,all P ＜ 0.05).The elevated expression level of miR-191 induced by NaAsO2 was significantly suppressed after transfected with miR-191 inhibitor (0.314 ± 0.094 vs 2.051 ± 0.371,P ＜ 0.05),which in turn up-regulated the protein expression of TIMP-3 (1.965 ± 0.277 vs 0.541 ± 0.183,P ＜ 0.05).Conclusion The expression level of miR-191 is elevated in response to NaAsO2 exposure,and miR-191 has subsequently suppressed the expression of TIMP-3,a potential target of miR-191.
ABSTRACT
OBJECTIVE: To explore the oxidation damage of sodium arsenite( NaAsO_2) drinking exposure on rat lung.METHODS: Thirty-two specific pathogen free healthy adult male SD rats were randomly divided into 4 groups: control group,low-,medium- and high-dose groups,with 8 rats in each group. The 3 arsenic exposure groups were intoxicated by NaAsO_2 with mass concentrations of 10. 00,100. 00 and 1 000. 00 μg / L in drinking water,respectively,while the control group was given ultrapure water for free drinking. All the rats were sacrificed after 28 days. The total number of white blood cells and the cell death rate in bronchoalveolar lavage fluid( BALF) were measured. The level of reactive oxygen species( ROS),nicotinamide adenine dinucleotide phosphate( NADPH),the antioxidant ability and the nitric oxide( NO) level in BALF were measured by the enzyme-linked immunosorbent assay. RESULTS: The total number of white blood cells in BALF in medium-dose group was higher than those of the control group and high-dose group( P < 0. 05).The cell death rate of BALF in high-dose group was higher than those of the other groups( P < 0. 01). The ROS levels in BALF of rat lung in 3 exposure groups were higher than that of the control group( P < 0. 01). The NADPH levels in medium- and high-dose groups were higher than those of the control group and low-dose group( P < 0. 05). The total antioxidant ability in medium- and high-dose groups were lower than that of the control group( P < 0. 01). The NO levels in medium- and high-dose groups were higher than that of the control group( P < 0. 05). CONCLUSION: The NaAsO_2 exposure in rats could lead to high expression of oxidase ROS by activating NADPH followed by increasing NO level,resulting in imbalanced organism oxidation and anti-oxidation system and causing oxidative stress injury in tissues.
ABSTRACT
Aims: To evaluate the protective effects of the aqueous extract of the fruit pulp of Adansonia digitata (AEFAD) in sodium arsenite (SA) and cyclophosphamide (CP) induced hepatotoxicity and clastogenicity in rats. Study Design/Methodology: Fifty four male Wistar rats were distributed into nine groups (A-I) of six animals each. Group A received distilled water and normal diet, Groups B received SA at 2.5 mg/kg body weight, Group C received CP at 10 mg/kg body weight, Groups D –I received the extract alone and with SA or CP. Results: A statistically significant (P <0.05) higher levels of: mean γGT, ALT and AST activities, number of micronucleated polychromatic erythrocytes (nMPCEs) scored in the bone marrow cells, proliferation of hepatic cells and lipid peroxidation were observed in rats exposed to (SA) or (CP) as compared with the control. Treatment with AEFAD along with SA or CP significantly (P <0.05) reduced the effects of the toxins on the above indices. Observations made with histological analysis of the liver sections revealed lesions ranging from general congestion, mild periportal cellular infiltration and hepatic necrosis to severe congestion in the treated groups. Conclusion: Findings from this study therefore reaffirmed the hepatoxicity and clastogenicity of SA and CP and revealed that AEFAD can ameliorate these toxicities in rats.
ABSTRACT
This work has been carried out in order to investigate the possible ameliorative effect of Coriander seeds methanolic extract (CSE) on sodium arsenite (As) – induced toxicity in albino rats. 2,3 Dimercaptosuccinic acid (DMSA) was used as a standerd chelating agent. The experiment lasted for 8 weeks and blood samples were withdrawn 4 and 8 weeks after As and other treatments administrations. As was used to induce hepatotoxicity in rats in a dose equivalent to 100 p.p.m . In-vivo studies using the different biochemical techniques employed in hepato-renal functions as well as liver apoptotic DNA and total RNA alterations proved that As caused a significant increase in parameters concerned to hepato-renal toxicity while treatment of CSE or DMSA caused an ameliorative effect on this toxicity. Administration of CSE and DMSA together along with arsenite proves the synergistic effects of these chelating agents on arsenite toxicity.
ABSTRACT
Objective To investigate the relationship between metabolites of sodium arsenite and sodium dihydrogen arsenate with related metabolic enzymes in kidney of male rats.Methods According to body mass,thirty-five male Wistar rats(body mass 150-190 g) were divided into 7 groups by random number table.Control group drank deionized water; the contents of iAsⅢ in low,medium and high arsenite groups and the contents of iAsv in low,medium and high of sodium dihydrogen arsenate groups were 2.2,6.7 and 20.0 mg/kg,respectively.After 3 months,kidneys were collected and stored at-80 C; high performance liquid chromatography and hydride genesis atomic fluorescence spectroscopy (HPLC-HGAFS) was used to determine the level of arsenic metabolites in kidney,and enzyme-linked immunosorbent assay was used to detect and analyze the content or the activity of metabolic enzymes,meanwhile correlation studies between the level of metabolites and the activity of metabolic enzymes were carried out.Results The differences of total arsenic (TAs),dimethyl arsenic acid (DMA),monomethyl arsenic acid (MMA) and methyl transferase enzyme activity in kidneys of rats between groups were statistically significant (F =1874.672,H =33.513,31.002,F =79.607,all P ＜ 0.01).The TAs[(526.52 ± 25.56),(1 654.00 ± 101.55),(1 904.24 ± 104.76)μg,/kg] and DMA[(323.20 + 16.13),(1 444.40 ± 113.81),(1 765.40 ± 104.39)μg/kg] of sodium arsenite in low,medium and high dose groups were higher than those of the corresponding sodium dihydrogen arsenate groups [(235.70 ± 6.23),(471.05 ± 18.32),(1 677.40 ± 83.29)μg/kg,and(0.00 ± 0.00),(1.75 ± 0.16),(410.50 ± 19.76)μg/kg,P ＜ 0.0024 or ＜ 0.05] ; the MMA[(4.02 + 0.86),(4.20 ± 0.65),(4.04 ± 0.80)μg/kg] of sodium arsenite in low,medium and high dose groups were lower than those of the corresponding sodium dihydrogen arsenate groups[(98.90 ± 9.59),(376.50 ± 15.41),(1 131.90 ± 74.26) μg/kg,all P＜ 0.05]; the methyl transferase enzyme activities[(7.80 ± 0.93),(5.55 ± 0.49),(3.56 ± 0.26)U/g] of sodium arsenite in low,medium and high dose groups were lower than those of the corresponding sodium dihydrogen arsenate group[(11.59 ± 0.93),(8.93 ± 0.88),(6.52 ± 1.04)U/g,all P ＜ 0.0024].The DMA of sodium arsenite in low,medium and high dose groups,the MMA of sodium dihydrogen arsenate in medium and high dose groups were positively correlated with those of TAs in each group(r =0.970,0.984,0.997,0.947,0.961,all P ＜ 0.05).Conclusions Effects of sodium arsenite and sodium dihydrogen arsenate on arsenic metobdites and related metabolic enzymes in kidney of rats are different.The function of sodium dihydrogen arsenate in promoting methyl transferase activity is stronger than that of sodium arsenite,which affects the amount and distribution of arsenic methylation metabolites in kidney.
ABSTRACT
Objective To investigate the influence of low-dose,long-term arsenite exposure on keratinocyte (HaCat) cell apoptosis and the expression of Bcl-2 and Bax proteins.Methods HaCat cells were exposed to different concentrations of NaAsO2[0(control group),0.05,0.10 μmol/L] for 15 weeks.A total of 10 000 cells in each group were measured to detect the level of apoptosis by flow cytometry.The protein expressions of Bcl-2 and Bax were determined by Western blotting method.Results The cell apoptosis rate was significantly different between groups (F =17.19,P ＜ 0.05).Compared with that of the control [(1.42 ± 0.13)％],the apoptosis rates of the 0.05 and 0.10 μmol/L groups [(1.23 ± 0.08)％ and (1.04 ± 0.06)％] decreased significantly (all P ＜ 0.05); the 0.10 μmol/L group was significantly lower than that of the 0.05 μmol/L group(P ＜ 0.05).The protein expressions of Bcl-2 and Bax were significantly different between groups (F=107.38,346.45,all P＜ 0.05).The protein expression of Bcl-2 of the 0.05 and 0.10 μmol/L groups were (143.89 ± 7.74)％ and (199.96 ± 12.18)％,respectively,which were significantly higher than that of the control group[(100.00 ± 1.45)％,all P ＜ 0.05] ; the 0.10 μmol/L group was significantly higher than that of the 0.05 μmol/L group (P ＜ 0.05).The protein expression of Bax of the 0.05 and 0.10 μmol/L groups were (70.78 ± 1.53)％ and (54.80 ± 1.34)％,respectively,which were markedly lower than that of the control group[(100.00 ± 3.09)％,all P ＜ 0.05]; the 0.10 μmoL/L group was significantly lower than that of the 0.05 μmol/L group(P ＜ 0.05).Conclusion Decreased level of apoptosis induced by low level and long-term arsenic exposure may be associated with increased expression of Bcl-2 protein and decreased expression of Bax protein.