The in vitro effects of Trolox on methylmercury-induced neurotoxicity

Methylmercury (MeHg), an environmental toxicant primarily found in fish and seafood poses a dilemma to both consumers and regulatory authorities given the nutritional benefits of fish consumption vs. possible adverse neurological damage caused by MeHg. The present study addresses whether supplementation with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), alters the neuro-oxidative effects of MeHg in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) were measured. The cellular mercury (Hg) content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). The amount of MeHg-induced ROS was significantly reduced (p < 0.05) after treatment with 50 μM Trolox in C6 glial cell line. However, treatment with Trolox did not induce any significant increase in GSH levels or MTT activity in either of the cell lines. In addition, treatment with Trolox did not induce any significant changes in intracellular MeHg levels. The MeHg and Trolox treated C6 glial cell line differed significantly (p < 0.05) from the B35 cell line for MTT, ROS and GSH activity. These findings provide experimental evidence that preincubation with Trolox prevents MeHg-induced ROS generation in C6 glial cell line by quenching of free radicals and not by changes in intracellular GSH or MeHg content.     

The in vitro effects of selenomethionine on methylmercury-induced neurotoxicity

Selenium (Se) has been reported to reduce the severity of MeHg-induced neurological deficits. Therefore, we investigated whether 24 h. preincubation or 50 min. coincubation with selenomethionine (SeMet) was effective in reducing methylmercury (MeHg)-induced cytotoxicity in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) was assessed. Measurement of GSH with the fluorescent indicator MCB-monochlorobimane indicated that in SeMet preincubated C6 cells, MeHg treatment resulted in a significant (p < 0.001) decrease in GSH levels as compared to coincubation group. Treatment with SeMet did not induce any significant changes in MTT activity in either of the cell lines as compared to the MeHg group. However, the amount of MeHg-induced ROS was significantly reduced (p < 0.001) after SeMet preincubation in both the cell lines. The intracellular Se content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). In both the cell lines the intracellular Se levels increased after pre- and coincubation with 20 and 50 μM SeMet. However, the preincubation group exhibited increased Se content in both the cell lines and varied (p < 0.001) from coincubation group. These differences in the Se content were maintained after 10 μM MeHg treatment for 50 min. In C6-gliomas, the cell associated-MeHg measurements using ¹⁴C-labeled MeHg indicated a significant increase (p < 0.001) in MeHg content in preincubated cells as compared to coincubated cells. These findings provide experimental evidence that preincubation with SeMet increases Se content in cells and prevents against increased MeHg-induced ROS generation.     

The use of fluorescence for detecting MeHg-induced ROS in cell cultures.

The effect of methylmercury (MeHg) on reactive oxygen species (ROS) induction in neural cell lines was measured by the fluorescent probe, chloro methyl derivative of di-chloro di-hydro fluoresceindiacetate (CMH2DCFDA). Three different MeHg concentrations (5, 10 and 25 microM) and time periods (30, 50 and 90 min) were studied in C6-glial and B35-neuronal cell lines. In addition, the relationship between MeHg-induced ROS and cell density (day 3 vs. day 4) was also explored. The 14C-labelled MeHg measurements were done to determine the cell associated-MeHg content. At 30 and 50 min exposure, a significant increase (p<0.05) in MeHg-induced ROS was observed at 10 and 25 microM MeHg for C6 cells and at 25 microM MeHg for B35 cells. However, the amount of ROS produced with 25 microM MeHg varied significantly (p<0.001) at different time periods. For both the cell lines, significant cell density dependent differences (p<0.05) were observed at 10 microM MeHg treatment for 50 min. MeHg treatments were associated with a concentration as well as cell-density dependent increase in cell associated-MeHg. These findings provide experimental evidence that special attention should be focused upon concentration, exposure time and cell density when assessing MeHg-induced ROS via fluorescence.     

Enhanced cell death effects of MAP kinase inhibitors in propyl gallate-treated lung cancer cells are related to increased ROS levels and GSH depletion

Propyl gallate (PG) has an anti-growth effect in lung cancer cells. The present study investigated the effects of mitogen-activated protein kinase (MAPK; MEK, JNK, and p38) inhibitors on PG-treated Calu-6 and A549 lung cancer cells in relation to cell death as well as reactive oxygen species (ROS) and glutathione (GSH) levels. PG induced cell death in both Calu-6 and A549 lung cancer cells at 24 h, which was accompanied by loss of mitochondrial membrane potential (MMP; ΔΨ_m). All of the tested MAPK inhibitors increased cell death in both PG-treated lung cancer cell lines. In particular, MEK inhibitor strongly enhanced cell death and MMP (ΔΨ_m) loss in PG-treated Calu-6 cells and p38 inhibitor had the same effects in A549 cells as well. PG increased ROS levels and caused GSH depletion in both cell lines at 24 h. MAPK inhibitors increased O₂^•- levels and GSH depletion in PG-treated Calu-6 cells, and JNK and p38 inhibitors increased ROS levels and GSH depletion in PG-treated A549 cells. In conclusion, MAPK inhibitors increased cell death in PG-treated Calu-6 and A549 lung cancer cells. Enhanced cell death and GSH depletion in Calu-6 cells caused by the MEK inhibitor were related to increased O₂^•- levels, and the effects of the p38 inhibitor in A549 cells were correlated with increased general ROS levels.     

Uptake and efflux of methylmercury in vitro: Comparison of transport mechanisms in C6, B35 and RBE4 cells

Methylmercury (MeHg) is a neurotoxicant which enters the brain and may cause permanent change. Thus, the properties of MeHg transport across cell membranes are a key factor in designing an appropriate model for MeHg neurotoxicity. This study uses cell cultures to examine the uptake and efflux mechanisms of methylmercury in C6 glioma, B35 neuroblastoma and rat brain endothelial (RBE4) cells. The cellular uptake and efflux of MeHg was investigated using ¹⁴C-labeled MeHg. The uptake of MeHg-chloride was temperature-independent while the uptake of MeHg-l-cysteine was temperature-dependent in all the three cell types. This indicates that uptake of MeHg-chloride is due to passive diffusion and uptake of MeHg-l-cysteine is due to a protein carrier. Substrates of the amino acid transport system L inhibited uptake of MeHg-l-cysteine in C6 and RBE4 cells, but not B35 cells, indicating a role for system L in MeHg-uptake in the former two. Probenecid, Na⁺-free medium, MeHg and several l-amino acids did not alter the efflux of MeHg from C6 and RBE4 cells. The amino acids l-cysteine and cystine however, increased the efflux. Both cysteine and cystine are important in the generation of glutathione (GSH), suggesting the involvement of GSH in MeHg efflux. HgCl₂ at low concentrations (0.5 and 1.0 μM) decreased the MeHg efflux and at high concentrations (5.0 and 10.0 μM) increased the efflux. This inhibiting effect of HgCl₂ at low concentrations is possibly due to binding to GSH while the effect of high HgCl₂ concentrations is attributed to disrupted membrane integrity, as measured by Trypan blue. This study demonstrates differing transport mechanisms of MeHg in the cell lines C6, B35 and RBE4.     

Role of glutathione in determining the differential sensitivity between the cortical and cerebellar regions towards mercury-induced oxidative stress

Certain discrete areas of the CNS exhibit enhanced sensitivity towards MeHg. To determine whether GSH is responsible for this particular sensitivity, we investigated its role in MeHg-induced oxidative insult in primary neuronal and astroglial cell cultures of both cerebellar and cortical origins. For this purpose, ROS and GSH were measured with the fluorescent indicators, CMH₂DCFDA and MCB. Cell associated-MeHg was measured with ¹⁴C-radiolabeled MeHg. The intracellular GSH content was modified by pretreatment with NAC or DEM. For each of the dependent variables (ROS, GSH, and MTT), there was an overall significant effect of cellular origin, MeHg and pretreatment in all the cell cultures. A trend towards significant interaction between origin × MeHg × pretreatment was observed only for the dependent variable, ROS (astrocytes p = 0.056; neurons p = 0.000). For GSH, a significant interaction between origin × MeHg was observed only in astrocytes (p = 0.030). The cerebellar cell cultures were more vulnerable (astrocytes_mean = 223.77; neurons_mean = 138.06) to ROS than the cortical cell cultures (astrocytes_mean = 125.18; neurons_mean = 107.91) for each of the tested treatments. The cell associated-MeHg increased when treated with DEM, and the cerebellar cultures varied significantly from the cortical cultures. Non-significant interactions between origin × MeHg × pretreatment for GSH did not explain the significant interactions responsible for the increased amount of ROS produced in these cultures. In summary, although GSH modulation influences MeHg-induced toxicity, the difference in the content of GSH in cortical and cerebellar cultures fails to account for the increased ROS production in cerebellar cultures. Hence, different approaches for the future studies regarding the mechanisms behind selectivity of MeHg have been discussed.     

Dimethyl fumarate induces necroptosis in colon cancer cells through GSH depletion/ROS increase/MAPKs activation pathway

Background and Purpose

Dimethyl fumarate (DMF) is a newly approved drug for the treatment of relapsing forms of multiple sclerosis and relapsing-remitting multiple sclerosis. Here, we investigated the effects of DMF and its metabolites mono-methylfumarate (MMF and methanol) on different gastrointestinal cancer cell lines and the underlying molecular mechanisms involved.

Experimental Approach

Cell viability was measured by the MTT or CCK8 assay. Protein expressions were measured by Western blot analysis. LDH release, live- and dead-cell staining, intracellular GSH levels, and mitochondrial membrane potential were examined by using commercial kits.

Key Results

DMF but not MMF induced cell necroptosis, as demonstrated by the pharmacological tool necrostatin-1, transmission electron microscopy, LDH and HMGB1 release in CT26 cells. The DMF-induced decrease in cellular GSH levels as well as cell viability and increase in reactive oxygen species (ROS) were inhibited by co-treatment with GSH and N-acetylcysteine (NAC) in CT26 cells. DMF activated JNK, p38 and ERK MAPKs in CT26 cells and JNK, p38 and ERK inhibitors partially reversed the DMF-induced decrease in cell viability. GSH or NAC treatment inhibited DMF-induced JNK, p38, and ERK activation in CT26 cells. DMF but not MMF increased autophagy responses in SGC-7901, HCT116, HT29 and CT26 cancer cells, but autophagy inhibition did not prevent the DMF-induced decrease in cell viability.

Conclusion and Implications

DMF but not its metabolite MMF induced necroptosis in colon cancer cells through a mechanism involving the depletion of GSH, an increase in ROS and activation of MAPKs.     

Propyl gallate decreases the proliferation of Calu-6 and A549 lung cancer cells via affecting reactive oxygen species and glutathione levels

Propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester, PG) has an anti-proliferative effect in various cells. In this study, Calu-6 and A549 lung cancer cells were used to examine the anti-proliferative effect of PG in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PG (100–1,600 μM) dose-dependently inhibited the proliferation of Calu-6 and A549 cells at 24 h, and PG at 800–1,600 μM strongly induced cell death in both cell lines. PG (800–1,600 μM) increased cellular metabolism in Calu-6 but not A549 cells at 4 h. PG either increased or decreased ROS levels, including O₂˙⁻ and ˙OH, depending on the incubation doses and times of 1 or 24 h. Even these effects differed between Calu-6 and A549 cell types. PG reduced the activity of superoxide dismutase (SOD) in Calu-6 cells, and it augmented the activity of catalase in A549 cells. PG dose-dependently increased the number of GSH depleted cells in both Calu-6 and A549 cells at 24 h. In addition, PG decreased GSH levels in both lung cancer cells at 1 h. Furthermore, diethyldithiocarbamate (DDC; an inhibitor of SOD) and 3-amino-1,2,4-triazole (AT; an inhibitor of catalase) differently affected cellular metabolism, ROS and GSH levels in PG-treated and PG-untreated Calu-6 and A549 cells at 1 h. In conclusion, PG dose-dependently decreased the proliferation of Calu-6 and A549 lung cancer cells, which was related to changes in ROS levels and the depletion of GSH.     

双氢青蒿素对人骨肉瘤细胞株143B增殖和凋亡的作用

目的观察双氢青蒿素(dihydroarteminin,DHA)对人骨肉瘤细胞143B的增殖和凋亡的影响以及可能的机制。方法体外培养人骨肉瘤细胞株143B;MTT比色法和克隆形成实验检测不同浓度DHA对骨肉瘤细胞存活与克隆形成能力的影响。Hoechst 33258染色法观察细胞凋亡的形态变化。构建β-Catenin荧光素酶报告基因(β-Catenin-Luc,p-Top)检测DHA作用143B后细胞内β-Catenin活性变化。不同浓度的DHA作用后,Western blot检测与细胞增殖(如PC-NA、Cyclin D1、c-Myc)、凋亡(如Bad、Bcl-2、Caspase-3)密切相关的标志物蛋白质表达变化。结果 DHA作用于143B细胞24 h后,MTT结果显示143B细胞的增殖活性受到明显抑制,且其克隆形成能力减弱(P<0.05),在DHA浓度达35μmol.L-1时,抑制效率最明显。Western blot结果显示PC-NA表达下调;而促凋亡蛋白Bad和Caspase-3表达上调,Bcl-2蛋白表达明显减弱。结论双氢青蒿素具有较明显的抑制人骨肉瘤细胞的增殖且促进其凋亡的作用,可能通过下调细胞增殖相关蛋白PCNA、Bcl-2和上调促凋亡蛋白Bad和Caspase-3,启动凋亡程序,致143B细胞发生凋亡。     

Treatment with p38 inhibitor intensifies the death of MG132-treated As4.1 juxtaglomerular cells via the enhancement of GSH depletion

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MG132 and/or MAPK inhibitors on As4.1 juxtaglomerular cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m) and activation of caspase-3 and -8. MG132 increased ROS levels, and GSH depleted cell numbers. The MEK inhibitor slightly reduced cell growth and caspase-3 activity in MG132-treated As4.1 cells and mildly increased MMP (ΔΨ_m) loss and O₂^?- level. However, it did not increase apoptosis and GSH depletion. The JNK inhibitor did not strongly influence cell growth, cell death, and GSH depletion by MG132, but increased caspase-3 activity, MMP (ΔΨ_m) loss, and O₂^?- level. Treatment with the p38 inhibitor magnified cell-growth inhibition and apoptosis by MG132. This agent also strongly increased caspase-8 activity, MMP (ΔΨ_m) loss, O₂^?- level, and GSH depletion. Conclusively, the p38 inhibitor strongly intensified cell death in MG132-treated As4.1 cells. The changes of GSH content by MG132 and/or MAPK inhibitors were closely related to the death of As4.1 cells.     

Harmonization of Mangiferin on methylmercury engendered mitochondrial dysfunction

Mangiferin (MGN), a C‐glucosylxanthone abundantly found in mango plants, was studied for its potential to ameliorate methylmercury (MeHg) induced mitochondrial damage in HepG2 (human hepatocarcinoma) cell line. Cell viability experiments performed using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐ diphenyltetrazolium bromide (MTT) showed protective property of MGN in annulling MeHg‐induced cytotoxicity. Conditioning the cells with optimal dose of MGN (50 µM) lowered MeHg‐induced oxidative stress, calcium influx/efflux, depletion of mitochondrial trans‐membrane potential and prevented mitochondrial fission as observed by decrease in Mitotracker red fluorescence, expression of pDRP1 (serine 616), and DRP1 levels. MGN pre‐treated cells demonstrated elevation in the activities of glutathione (GSH), Glutathione‐S‐transferase (GST), Glutathione peroxidase (GPx), Glutathione reductase (GR), reduced levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) and mitochondrial electron transport chain (ETC) enzyme complexes. In addition, the anti‐apoptotic effect of MGN was clearly indicated by the reduction in MeHg‐induced apoptotic cells analyzed by flowcytometric analysis after Annexin V‐FITC/propidium iodide staining. In conclusion, the present work demonstrates the ability of a dietary polyphenol, MGN to ameliorate MeHg‐mediated mitochondrial dysfunction in human hepatic cells in vitro. This hepatoprotective potential may be attributed predominantly to the free radical scavenging/antioxidant property of MGN, by facilitating the balancing of cellular Ca²⁺ ions, maintenance of redox homeostasis and intracellular antioxidant activities, ultimately preserving the mitochondrial function and cell viability after MeHg intoxication. As MeHg intoxication occurs over a period of time, continuous consumption of such dietary compounds may prove to be very useful in promoting human health. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 630–644, 2017.     

MG132, a proteasome inhibitor-induced calf pulmonary arterial endothelial cell growth and death,are changed by MAPK inhibitors

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK inhibitors on MG132-treated calf pulmonary artery endothelial cells (CPAECs) in relation to cell death, ROS, and glutathione (GSH). MG132 inhibited the growth of CPAEC and also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). MG132 increased ROS levels and GSH-depleted cell numbers in CPAEC. Treatment with MAPK (MEK, JNK, and p38) inhibitors showed a slight enhancement of cell-growth inhibition by MG132. All the MAPK inhibitors decreased cell death by MG132. Especially, the JNK inhibitor showed a strong effect. They all did not affect ROS levels and GSH depletion in MG132-treated CPAEC, but increased ROS and GSH levels in MG132-untreated CPAEC. In conclusion, MG132 induced apoptosis in CPAEC, which was accompanied by ROS increase and GSH depletion. The changes of MG132-induced CPAEC growth inhibition and death by MAPK inhibitors were not tightly correlated to ROS and GSH levels.     

Mitogen-activated protein kinase inhibitors differently affect the growth inhibition and death of a proteasome inhibitor, MG132-treated human pulmonary fibroblast cells

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor can induce growth inhibition and death in lung cancer or normal cells. However, little is known about relationship between proteasome inhibition and mitogen-activated protein kinase (MAPK) signaling in normal lung cells. Thus, in the present study, we investigated the effects of MAPK inhibitors on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition, cell death, reactive oxygen species (ROS) and glutathione (GSH). Treatment with 15 μM MG132 increased ROS levels including mitochondrial O(2?)(-) and GSH depleted cell numbers in HPF cells at 24 hours. MAP kinase or ERK kinase (MEK) inhibitor did not significantly affect cell growth inhibition, cell death, the loss of mitochondrial membrane potential (MMP; ΔΨ(m)), ROS level and GSH depletion in MG132-treated HPF cells. c-Jun N-terminal kinase (JNK) inhibitor attenuated the growth inhibition and death by MG132. This inhibitor also significantly decreased O(2?)(-) level in MG132-treated HPF cells. Although p38 inhibitor slightly enhanced HPF cell growth inhibition by MG132, this inhibitor and siRNA prevented HPF cell death induced by MG132. p38 inhibitor also attenuated d O(2?)(-) level and GSH depletion. Moreover, extracellular signal regulated kinase (ERK), JNK or p38 siRNA did not strongly affect ROS levels in MG132-treated HPF cells. ERK and JNK siRNAs decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. In conclusion, MAPK inhibitors differently affected the growth inhibition and death of MG132-treated HPF cells. Especially, p38 inhibitor attenuated HPF cell death by MG132, which was in part related to changes in ROS and GSH levels.     

Ferric iron increases ROS formation, modulates cell growth and enhances genotoxic damage by 4-hydroxynonenal in human colon tumor cells.

Iron is a relevant risk factor for colorectal cancer due to its genotoxic properties. Here we hypothesised that iron-overload causes other toxic effects, which contribute to carcinogenesis. For this, we investigated formation of reactive oxygen species (ROS), DNA repair, cell growth and glutathione (GSH) in human colon tumor cells (HT29 clone 19A) treated with ferric nitrilotriacetate (Fe-NTA, 0-2000 microM). Intracellular formation of ROS was analysed with the peroxide-labile fluorescent dye carboxy-dichlorodihydrofluorescine-diacetate. DNA repair, reflected as the persistency of DNA damage induced by selected genotoxins, was determined with the Comet assay. Cell growth and GSH were measured by fluorimetrical analysis. Key findings were that ROS formation increased with time (1000 microM Fe-NTA, p < 0.001). DNA damage was largely repaired after 120 min, but was not affected by 10 microM Fe-NTA. In contrast, 10 microM Fe-NTA significantly increased DNA damage induced by 4-hydroxynonenal. Doses of 25 microM Fe-NTA increased cell growth (p < 0.05), whereas high concentrations (2000 microM) resulted in growth arrest (p < 0.05), that was accompanied by increased GSH levels (p < 0.01). In conclusion, high concentrations of Fe-NTA caused cellular effects, which reflect a stress response, and resulted in formation of ROS. Carcinogenic risks from ferric iron could be derived also from lower concentrations, which enhance tumor cell growth and cause progenotoxic effects.     

2,4-dinitrophenol induces G1 phase arrest and apoptosis in human pulmonary adenocarcinoma Calu-6 cells.

2,4-dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in the mitochondria. Here, we investigated the effect of DNP on the growth of Calu-6 lung cancer cells in view of cell cycle, apoptosis, ROS production and GSH content. DNP dose-dependently decreased cell viability at 72 h (EC50 of about 200 microM) as measured by a MTT assay. The lower doses of DNP induced a G1 arrest of the cell cycle in Calu-6 cells. Analysis of the cell cycle regulatory proteins demonstrated that DNP decreased the steady-state levels of cyclin proteins and cyclin dependent kinase (CDK), but increased the protein levels of cyclin dependent kinase inhibitor (CDKI) p27. DNP also caused a marked increase in apoptosis, as evidenced by DNA fragmentation (sub-G1 DNA content), DAPI staining, the loss of mitochondrial membrane potential (DeltaPsim), externalization of phosphatidylserine (PS). In addition, DNP-treated cells significantly increased the intracellular H2O2 and O2.- levels. All of caspase inhibitors could markedly rescue Calu-6 cells from DNP-induced cell death and only pan-caspase inhibitor, Z-VAD-FMK, could slightly prevent the loss of mitochondrial membrane potential (DeltaPsim). However, none of the caspase inhibitors reduced the increased H2O2 levels, but the increased O2.- levels was slightly attenuated by pan-caspase inhibitor. In addition, the depletion of GSH content in DNP-treated cells was prevented by all of caspase inhibitors. In conclusion, DNP, which induced ROS and reduced GSH content, inhibited the growth of Calu-6 cells via cell cycle arrest at G1 phase and apoptosis.     

Alpha‐lipoic acid reduces methylmercury‐induced neuronal injury in rat cerebral cortex via antioxidation pathways

Methylmercury (MeHg), an extremely dangerous environmental pollutant, accumulating preferentially in central nervous system, causes a series of cytotoxic effects. The present study explored the mechanisms which contribute to MeHg‐induced neurotoxicity focusing on the oxidative stress in rat cerebral cortex. In addition, the protective effects of alpha‐lipoic acid (LA), a potent antioxidant on MeHg‐mediated neuronal injury, was also investigated in current study. A MeHg poisoning model was established as 64 rats randomly divided into 4 groups of which saline control group, MeHg‐treated groups (4 and 12 μmol kg^?1), and LA pretreatment (35 μmol kg^?1) group, respectively. After administration of 12 μmol kg^?1 MeHg for 4 weeks, it was found that obvious pathological changes and apoptosis in neuronal cells. Meanwhile, total Hg levels elevated significantly, superoxide dismutase (SOD) and gluthathione peroxidase (GSH‐Px) activities were inhibited, and ROS formation elevated, which might be critical to aggravate oxidative stress in cerebral cortex. In addition, NF‐E2‐related factor 2 (Nrf2) pathways were activated, as heme oxygenase‐1 (HO‐1) and γ‐glutamylcysteine synthetase heavy subunit (γ‐GCSh) expressions were up‐regulated obviously by MeHg exposure. Moreover, activities of Na⁺‐K⁺‐ATPase and Ca²⁺‐ATPase were inhibited, leading to intracellular calcium (Ca²⁺) overload. LA pre‐treatment partially reduced MeHg neurotoxic effects via anti‐oxidation pathways. In conclusion, these findings clearly indicated that MeHg aggravated oxidative stress and Ca²⁺ overload in cerebral cortex. LA possesses the ability to prevent MeHg neurotoxicity through its anti‐oxidative properties. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 931–943, 2017.     

DHA诱导胃癌细胞SGC-7901凋亡及其作用机制研究

目的 探究二十二碳六烯酸(DHA)对人胃癌细胞SGC-7901生长的影响及诱导细胞凋亡的作用机制.方法 MTT法测定细胞存活率.Hoechst染色法进行细胞形态学观察.流式细胞仪检测细胞凋亡和活性氧(ROS)的变化,硫代巴比妥钠比色法(TBA法)测定细胞脂质过氧化产物丙二醛(MDA)含量变化,二硫代二硝基苯甲酸(DNTB)比色法测定谷胱甘肽(GSH)含量.结果 MTT和流式细胞仪分析结果显示.细胞增殖率随DHA处理浓度的递增逐渐下降,呈现明显剂量效应关系.形态学观察显示凋亡细胞细胞质凝聚、浓染,凋亡小体出现.60.80,100/μmol·L-1DHA作用细胞24 h后,MDA含量显著高于对照组(P<0.05);GSH含量下降(P<0.05).80μmol·L-1DHA处理细胞5 h后,ROS出现峰值,且抗氧化剂NAC可以减弱ROS的变化.结论 DHA可以诱导胃癌细胞SOC-7901凋亡,脂质过氧化水平和RoS升高可能是DHA诱导细胞凋亡的重要机制.     

Nanotoxicity of pure silica mediated through oxidant generation rather than glutathione depletion in human lung epithelial cells

Though, oxidative stress has been implicated in silica nanoparticles induced toxicity both in vitro and in vivo, but no similarities exist regarding dose–response relationship. This discrepancy may, partly, be due to associated impurities of trace metals that may present in varying amounts. Here, cytotoxicity and oxidative stress parameters of two sizes (10 nm and 80 nm) of pure silica nanoparticles was determined in human lung epithelial cells (A549 cells). Both sizes of silica nanoparticles induced dose-dependent cytotoxicity as measured by MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of reactive oxygen species (ROS) generation, and membrane lipid peroxidation (LPO). However, both sizes of silica nanoparticles had little effect on intracellular glutathione (GSH) level and the activities of glutathione metabolizing enzymes; glutathione reductase (GR) and glutathione peroxidase (GPx). Buthionine-[S,R]-sulfoximine (BSO) plus silica nanoparticles did not result in significant GSH depletion than that caused by BSO alone nor N-acetyl cysteine (NAC) afforded significant protection from ROS and LPO induced by silica nanoparticles. The rather unaltered level of GSH is also supported by finding no appreciable alteration in the level of GR and GPx. Our data suggest that the silica nanoparticles exert toxicity in A549 cells through the oxidant generation (ROS and LPO) rather than the depletion of GSH.     

p21(WAF1/CIP1) inhibits cell cycle progression but not G2/M-phase transition following methylmercury exposure

Methylmercury (MeHg) is an environmentally prevalent organometal that is particularly toxic to the developing central nervous system (CNS). Prenatal MeHg exposure is associated with reduced brain size and weight and a reduced number of neurons, which have been associated with impaired cell proliferation. We evaluate the role of p21, a cell cycle protein involved in the G1- and G2-phase checkpoint control, in the cell cycle inhibition induced by MeHg. Primary mouse embryonic fibroblasts (MEFs) of different p21 genotypes (wild-type, heterozygous, and null) were isolated at day 14 of gestation and treated at passages 4-6 with either 0, 2, 4, or 6 microM MeHg or 50 nM colchicine for 24 h. Changes in cell cycle distribution after continuous toxicant treatment were analyzed by DNA content-based flow cytometry using DAPI. MeHg induced an increase in the proportion of cells in G2/M at 2 and 4 microM MeHg (p < or = 0.05) irrespective of p21 genotype. Effects of MeHg on cell cycle progression were subsequently evaluated using BrdU-Hoechst flow cytometric analysis. Inhibition of cell cycle progression was observed in all p21 genotypes after continuous exposure to MeHg for 24 and 48 h. p21 null (-/-) cells reached the second-round G1 at a higher fraction compared to the wild type (+/+) and heterozygous (+/-) cells (p < or = 0.05). These data support previous observations that MeHg inhibits cell cycle progression through delayed G2/M transition. Whereas the G2/M accumulation induced by MeHg was independent of p21 status, a greater proportion of p21(-/-) cells were able to complete one round of cell division in the presence of MeHg compared to p21(+/-) or p21(+/+) cells. These data suggest a role for p21 in retarding cell cycle progression, but not mitotic inhibition, following exposure to MeHg.     

Enhancement of gallic acid-induced human pulmonary fibroblast cell death by N-acetyl cysteine and L-buthionine sulfoximine

Gallic acid (GA) has various biological properties including anti-cancer effect. However, little is known about the toxicological effect of GA in primary normal cells. Here, we evaluated the effects of GA on human pulmonary fibroblast (HPF) cells in relation to reactive oxygen species (ROS) and glutathione (GSH). GA inhibited the growth of HPF cells at 24 hours in a dose-dependent manner. GA also induced HPF cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). GA increased ROS levels including O(2)(?-) and GSH-depleted cell numbers in HPF cells at 24 hours. Treatment with 2 mM N-acetyl-cysteine (NAC) intensified growth inhibition and death in GA-treated HPF cells. NAC decreased ROS levels and increased GSH depletion in these cells. Treatment with 10 μM L-buthionine sulfoximine (BSO) also enhanced growth inhibition and death in GA-treated HPF cells. BSO increased ROS levels and GSH depletion in these cells. In conclusion, GA-induced HPF cell death was accompanied by ROS increase and GSH depletion. The changes of ROS and GSH levels by NAC and BSO appeared to affect cell growth and death in GA-treated HPF cells.