Oxidative stress-induced cytotoxic and genotoxic effects of nano-sized titanium dioxide particles in human HaCaT keratinocytes

Since nano-sized particles (NPs) are increasingly used in various fields of innovative biomedicine and industrial technologies, it is of importance to identify their potential human health risk. We investigated whether ROS-induced mitochondrial DNA damage is the mode of action of titanium dioxide-NPs (TiO2-NPs; ≤20 nm) to induce cytotoxic and genotoxic effects in human HaCaT keratinocytes in vitro. We showed that TiO2-NPs accumulate at the cell surface and are taken up by endocytosis. Micronucleus (MN) formation was found to be significantly maximal increased 24 h after treatment with 10 μg/ml and 48 h after treatment with 5 μg/ml TiO2-NPs about 1.8-fold respectively 2.2-fold of control. Mitochondrial DNA damage measured as "common deletion" was observed to be significantly 14-fold increased 72 h after treatment with 10 μg/ml TiO2-NPs when compared to control. Four hours after treatment with 5 and 50 μg/ml TiO2-NPs the level of ROS in HaCaT cells was found to be significantly increased about 7.5-fold respectively 16.7-fold of control. In conclusion, for the first time we demonstrate the induction of the mitochondrial "common deletion" in HaCaT cells following exposure to TiO2-NPs, which strongly suggests a ROS-mediated cytotoxic and genotoxic potential of NPs. However, the effects of the modification of TiO2-NPs, such as agglomeration, size distribution pattern and exposure time have to be further critically examined.     

Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells

Titanium dioxide nanoparticles (TiO(2)-NPs) are produced in large quantities, raising concerns about their impact for human health. The aim of this study was to deeply characterize TiO(2)-NPs genotoxic potential to lung cells, and to link genotoxicity to physicochemical characteristics, e.g., size, specific surface area, crystalline phase. A549 cells were exposed to a panel of TiO(2)-NPs with diameters ranging from 12 to 140 nm, either anatase or rutile. A set of complementary techniques (comet and micronucleus assays, gamma-H2AX immunostaining, 8-oxoGuanine analysis, H2-DCFDA, glutathione content, antioxidant enzymes activities) allowed us to demonstrate that small and spherical TiO(2)-NPs, both anatase and rutile, induce single-strand breaks and oxidative lesions to DNA, together with a general oxidative stress. Additionally we show that these NPs impair cell ability to repair DNA, by inactivation of both NER and BER pathways. This study thus confirms the genotoxic potential of TiO(2)-NPs, which may preclude their mutagenicity and carcinogenicity.     

Titanium dioxide nanoparticles induced intracellular calcium homeostasis modification in primary human keratinocytes. Towards an in vitro explanation of titanium dioxide nanoparticles toxicity

Deciphering the molecular basis of toxicology mechanism induced by nanoparticles (NPs) remains an essential challenge. Ion Beam Analysis (IBA) was applied in combination with Transmission Electron Microscopy and Confocal Microscopy to analyze human keratinocytes exposed to TiO(2)-NPs. Investigating chemical elemental distributions using IBA gives rise to a fine quantification of the TiO(2)-NPs uptake within a cell and to the determination of the intracellular chemical modifications after TiO(2)-NPs internalization. In addition, fluorescent dye-modified TiO(2)-NPs have been synthesized to allow their detection, precise quantification and tracking in vitro. The internalization of these TiO(2)-NPs altered the calcium homeostasis and induced a decrease in cell proliferation associated with an early keratinocyte differentiation, without any indication of cell death. Additionally, the relation between the surface chemistry of the TiO(2)-NPs and their in vitro toxicity is clearly established and emphasizes the importance of the calcium homeostasis alteration in response to the presence of TiO(2)-NPs.     

Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents

Titanium dioxide nanoparticles (TiO₂-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1–50?μg/mL TiO₂-NPs, 86% anatase/14% rutile, 24?±?6?nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO₂-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO₂-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO₂-NPs.     

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO₂ NPs were used to compare the effects. Exposure to 32?μg/mL ZnO NPs (p?2 NPs (p?>?0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO₄ induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO₂ NP exposure (p?>?0.05). The presence of 250?nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p?p?>?0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p?>?0.05) except neutral red uptake assay (p?相似文献   

Oxidative stress induced by pure and iron-doped amorphous silica nanoparticles in subtoxic conditions

Amorphous silica nanoparticles (SiO?-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO?-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO?-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO?-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40-50 μg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO?-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 μg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.     

Oxidative damage and OGG1 expression induced by a combined effect of titanium dioxide nanoparticles and lead acetate in human hepatocytes

Titanium dioxide (TiO(2)) is a widely used nanomaterial that can cause biological damage through oxidative stress. At low concentrations, TiO(2) can interact with lead acetate (PbAc) to produce different toxic responses, compared with TiO(2) or PbAc alone. In this study, we utilized the following as indicators of toxic responses in human embryo hepatocytes (L02): reactive oxygen species (ROS), reduced glutathione (GSH), superoxide dismutase (SOD), and the DNA adducts 8-hydroxydeoxyguanosine (8-OHdG) and 8-oxoguanine DNA glycosylase homolog 1 (OGG1). These were used to evaluate the oxidative stress of TiO(2) (at 0.001, 0.01, 0.1, 1, and 10 μg mL(-1)) mixed with PbAc (1 μg mL(-1)) on L02 cells without photoactivation. Compared with the negative control (1‰ dimethyl sulfoxide), TiO(2) mixed with PbAc induced increased release of ROS (at 0.001, 0.01, 0.1, 1, 10 μg mL(-1) TiO(2)), intracellular SOD activity (at 0.1 and 0.01 μg mL(-1) TiO(2)), GSH levels (at 0.01-1 μg mL(-1) TiO(2)), 8-OHdG levels (at 1 and 10 μg mL(-1) TiO(2)), OGG1 expression (at 0.001-1 μg mL(-1) TiO(2)), and cytotoxicity (at 0.1, 1, and 10 μg mL(-1) TiO(2)) in L02 cells. There were no significant changes in ROS, GSH, SOD, 8-OHdG, or OGG1 levels when L02 cells were treated with TiO(2) alone or PbAc alone. These findings indicate that TiO(2) and PbAc in combination induce cytotoxicity and oxidative stress in L02 cells in the absence of photoactivation.     

TiO2 nanoparticles induce endothelial cell activation in a pneumocyte–endothelial co-culture model

The effects of particulate matter (PM) on endothelial cells have been evaluated in vitro by exposing isolated endothelial cells to different types of PM. Although some of the findings from these experiments have been corroborated by in vivo studies, an in vitro model that assesses the interaction among different cell types is necessary to achieve more realistic assays. We developed an in vitro model that mimics the alveolar–capillary interface, and we challenged the model using TiO₂ nanoparticles (TiO₂-NPs). Human umbilical endothelial cells (HUVECs) were cultured on the basolateral side of a membrane and pneumocytes (A549) on the apical side. Confluent co-cultures were exposed on the apical side to 10 μg/cm² of TiO₂-NPs or 10 ng/mL of TNFα for 24 h. Unexposed cultures were used as negative controls. We evaluated monocyte adhesion to HUVECs, adhesion molecule expression, nitric oxide concentration and proinflammatory cytokine release. The TiO₂-NPs added to the pneumocytes induced a 3- to 4-fold increase in monocyte adhesion to the HUVECs and significant increases in the expression of adhesion molecules (4-fold for P-selectin at 8 h, and about 8- and 10-fold for E-selectin, ICAM-1, VCAM-1 and PECAM-1 at 24 h). Nitric oxide production also increased significantly (2-fold). These results indicate that exposing pneumocytes to TiO₂-NPs causes endothelial cell activation.     

Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H2O2-induced DNA damage in cultured human leukocytes

Essential oil (EO) of Alpinia zerumbet leaves, at non-toxic concentrations (50–300 μg/mL), did not induce genotoxicity in human leukocytes. However, at the highest concentration (500 μg/mL) tested caused a reduction in cell proliferation and viability, and an increase in DNA damage. Moreover, in vivo experiments showed that EO (400 mg/kg) did not exert mutagenicity on peripheral blood cells and bone marrow in mice. In DPPH test, EO showed scavenging effects against DPPH radicals, and other free radicals (determination of intracellular GSH and lipid peroxidation assays). Furthermore, EO was able to reduce the intracellular levels of ROS, and prevented leukocytes DNA against oxidative damage. The ability of EO to reduce H₂O₂ toxicity was observed only when cells were treated with EO during and after exposure to H₂O₂. With the co- and post-treatment procedures, EO decreased the frequency of apoptotic and micronucleated leukocytes as well DNA strand breaks. However, a synergistic effect was observed in cultures exposed to 500 μg/mL EO. In conclusion, EO at concentrations up to 300 μg/mL or doses up to 400 mg/kg are not mutagenic in leukocytes and in mice, but do have antioxidative and protective effects against the cytotoxicity and clastogenesis induced by H₂O₂.     

The in vivo underlying mechanism for recovery response formation in nano-titanium dioxide exposed Caenorhabditis elegans after transfer to the normal condition

So far, we still know little about mechanism for recovery response of engineered nanomaterials (ENMs). Here we used Caenorhabditis elegans to investigate recovery responses of titanium dioxide nanoparticles (TiO₂-NPs) exposed animals and the underlying mechanism. After acute exposure to TiO₂-NPs (100 mg/L), endpoints including defecation and permeable state of intestinal barrier of exposed nematodes returned to control levels; however, after prolonged exposure to TiO₂-NPs (100 μg/L), endpoints of exposed nematodes could not be recovered to control levels under the normal condition. After prolonged exposure to TiO₂-NPs, nematodes exhibited severe deficits in development of intestinal barrier and AVL and DVB neurons controlling defecation; however, after acute exposure to TiO₂-NPs, nematodes had normal developmental state of intestinal barrier and AVL and DVB neurons. Our results imply that developmental states of intestinal barrier and AVL and DVB neurons may serve as a pivotal determinant for recovery response in TiO₂-NPs exposed nematodes.From the Clinical EditorThis basic science study investigates the recovery response to TiO₂ nanoparticles in a nematode model, and concludes that developmental states of the intestinal barrier and AVL and DVB neurons likely serve as determinants for recovery following TiO2-NP exposure.     

Investigations on the inflammatory and genotoxic lung effects of two types of titanium dioxide: untreated and surface treated

TiO(2) is considered to be toxicologically inert, at least under nonoverload conditions. To study if there are differences in lung effects of surface treated or untreated TiO(2) we investigated the inflammatory and genotoxic lung effects of two types of commercially available TiO(2) at low doses relevant to the working environment. Rats were exposed by instillation to a single dose of 0.15, 0.3, 0.6, and 1.2 mg of TiO(2) P25 (untreated, hydrophilic surface) or TiO(2) T805 (silanized, hydrophobic surface) particles, suspended in 0.2 ml of physiological saline supplemented with 0.25% lecithin. As control, animals were instilled with the vehicle medium only or with a single dose of 0.6 mg quartz DQ12. At days 3, 21, and 90 after instillation bronchoalveolar lavage was performed and inflammatory signs such as cells, protein, tumor necrosis factor-alpha, fibronectin, and surfactant phospholipids were determined. Additionally, 8 microm frozen sections of the left lobe of the lung were cut and stored at -80 degrees C. The sections were used for immunohistochemical detection of 8-oxoguanine (8-oxoGua) by a polyclonal antibody in the DNA of individual lung cells. In the quartz-exposed animals a strong progression in the lung inflammatory response was observed. Ninety days after exposure a significant increase in the amount of 8-oxoGua in DNA of lung cells was detected. In contrast, animals exposed to TiO(2) P25 or TiO(2) T805 showed no signs of inflammation. The amount of 8-oxoGua as a marker of DNA damage was at the level of control. The results indicate that both types of TiO(2) are inert at applicated doses.     

Size influences the cytotoxicity of poly (lactic-co-glycolic acid) (PLGA) and titanium dioxide (TiO2) nanoparticles

The aim of this study is to uncover the size influence of poly (lactic-co-glycolic acid) (PLGA) and titanium dioxide (TiO₂) nanoparticles on their potential cytotoxicity. PLGA and TiO₂ nanoparticles of three different sizes were thoroughly characterized before in vitro cytotoxic tests which included viability, generation of reactive oxygen species (ROS), mitochondrial depolarization, integrity of plasma membrane, intracellular calcium influx and cytokine release. Size-dependent cytotoxic effect was observed in both RAW264.7 cells and BEAS-2B cells after cells were incubated with PLGA or TiO₂ nanoparticles for 24 h. Although PLGA nanoparticles did not trigger significantly lethal toxicity up to a concentration of 300 μg/ml, the TNF-α release after the stimulation of PLGA nanoparticles should not be ignored especially in clinical applications. Relatively more toxic TiO₂ nanoparticles triggered cell death, ROS generation, mitochondrial depolarization, plasma membrane damage, intracellular calcium concentration increase and size-dependent TNF-α release, especially at a concentration higher than 100 μg/ml. These cytotoxic effects could be due to the size-dependent interaction between nanoparticles and biomolecules, as smaller particles tend to adsorb more biomolecules. In summary, we demonstrated that the ability of protein adsorption could be an important paradigm to predict the in vitro cytotoxicity of nanoparticles, especially for low toxic nanomaterials such as PLGA and TiO₂ nanoparticles.     

Protective effect of polypeptides from larva of housefly (Musca domestica) on hydrogen peroxide-induced oxidative damage in HepG2 cells

Housefly (Musca domestica) is an important medical insect and its larva is an ideal high protein food source. We isolated from housefly larvae the polypeptides hydrolyzed by neutral protease (PHNP), and investigated the protective effect of PHNP on hydrogen peroxide (H₂O₂)-induced oxidative damage in HepG2 cells. Cells exposed to H₂O₂ showed a marked decrease in proliferation and intracellular superoxide dismutase (SOD) activity, and a significant increase in reactive oxygen species (ROS) level and malondialdehyde (MDA) content. H₂O₂ also caused apoptosis and mitochondrial dysfunction including mitochondrial fragmentation and the loss of mitochondrial membrane potential. Pretreatment with PHNP at concentrations of 2.5, 5, 10 μg/mL blocked these H₂O₂-induced cellular events in a dose-dependent manner. The effect of PHNP at 10 μg/mL is equal to that of ascorbic acid at 10 μM. In summary, PHNP has a protective effect against H₂O₂-induced oxidative injury in cells due to its ability to decrease intracellular ROS and elevate antioxidant enzyme activities.     

Assessment of DNA damage,cytotoxicity, and apoptosis in human hepatoma (HepG2) cells after flurochloridone herbicide exposure

In vitro effects of flurochloridone (FLC) and its formulations Twin Pack Gold® [25% active ingredient (a.i.)] and Rainbow® (25% a.i.) were evaluated in HepG2 cells. Whereas cytokinesis-blocked micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were employed for genotoxicity, MTT, neutral red, and apoptosis detections were used for cytotoxicity evaluation. Activities were tested within the concentration range of 0.25–15 μg/ml FLC. Results demonstrated that neither FLC nor Rainbow® was able to induce MNs. On the other hand, 5 μg/ml Twin Pack Gold® only increased MN frequency. Furthermore, 10 and 15 μg/ml of both formulations resulted in cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. A marked increase in the genetic damage index was observed after treatment with all compounds. SCGE assay appeared to be more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of FLC than did MN. Our results reveal that FLC and its two formulations trigger apoptosis on HepG2 cells. The results represent the first experimental evidence of the in vitro apoptogenic role exerted on mammalian cells by FLC and the FLC-based formulations Rainbow® and Twin Pack Gold®, at least on HepG2 cells.     

Propoxur-induced oxidative DNA damage in human peripheral blood mononuclear cells: protective effects of curcumin and α-tocopherol

The present study enumerates the attenuating effects of curcumin and α-tocopherol against propoxur induced oxidative DNA damage in human peripheral blood mononuclear cells (PBMC). Cultured cells were isolated from peripheral blood of healthy volunteers, and were exposed to varying concentrations of propoxur (0–21?μg/ml) for 6, 12, and 24?h, and in combination with curcumin (9.2?μg/ml) or α-tocopherol (4.3?μg/ml) or both. Cytotoxic effect of propoxur was examined by MTT assay. The role of oxidative stress beneath the cytotoxicity of propoxur was evaluated by the measurement of reduced glutathione (GSH), malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) levels in cell lysate. A concentration-dependent cell death, depletion of GSH, an increase in the level of both MDA and 8-OH-dG were observed. Co-treatment with curcumin or α-tocopherol significantly attenuates depleted GSH, decrease in MDA and 8-OH-dG levels in propoxur exposed cells (p?相似文献   

Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells

Serious concerns have been expressed about potential risks of engineered nanoparticles. Regulatory health risk assessment of such particles has become mandatory for the safe use of nanomaterials in consumer products and medicines; including the potential effects on reproduction and fertility, are relevant for this risk evaluation. In this study, we examined effects of silver particles of nano- (20nm) and submicron- (200nm) size, and titanium dioxide nanoparticles (TiO(2)-NPs; 21nm), with emphasis on reproductive cellular- and genotoxicity. Ntera2 (NT2, human testicular embryonic carcinoma cell line), and primary testicular cells from C57BL6 mice of wild type (WT) and 8-oxoguanine DNA glycosylase knock-out (KO, mOgg1(-/-)) genotype were exposed to the particles. The latter mimics the repair status of human testicular cells vs oxidative damage and is thus a suitable model for human male reproductive toxicity studies. The results suggest that silver nano- and submicron-particles (AgNPs) are more cytotoxic and cytostatic compared to TiO(2)-NPs, causing apoptosis, necrosis and decreased proliferation in a concentration- and time-dependent manner. The 200nm AgNPs in particular appeared to cause a concentration-dependent increase in DNA-strand breaks in NT2 cells, whereas the latter response did not seem to occur with respect to oxidative purine base damage analysed with any of the particles tested.     

Genotoxicity and oxidative stress of the mutagenic compounds formed in fumes of heated soybean oil,sunflower oil and lard

This study was to investigate the genotoxicity and cytotoxicity of the oil fumes formed from heating three common commercial cooking oils (soybean oil, sunflower oil, and lard) on human lung carcinoma pulmonary type II-like epithelium cell (A-549 cell). The major alkenal mutagenic compounds (trans-trans-2,4-decadienal, t-t-2,4-DDE; trans-trans-2,4-nonadienal, t-t-2,4-NDE; trans-2-decenal, t-2-DCA and trans-2-undecenal, t-2-UDA) contained in three oil fumes and their effects on the induction of reactive oxygen species (ROS) were also studied. It was found that the most potent mutagenic compound (t-t-2,4-DDE) of oil fumes was 66.4, 35.9 and 40.3 μg/g in soybean oil, sunflower oil and lard, respectively. The results indicated that the methanolic extracts of oil fumes could apparently lead to cytotoxicity and oxidative DNA damage. Glutathione (GSH) contents and the activities of antioxidant enzymes such as GSH reductase, and GSH S-transferase were adversely reduced by the methanolic extracts of oil fumes. When human A-549 cells were exposed to the methanolic extracts of oil fumes for 30 min, there was an increase in the formation of intracellular ROS, which was determined by dichlorofluorescein assay. Moreover, the methanolic extracts of oil fumes caused significant (p < 0.05) oxidative damage through the 8-hydroxy-2′-deoxyguanosine formation in A-549 cells at the concentrations from 50 to 200 μg/ml. These results demonstrated that the DNA damage in A-549 cells, induced by cooking oil fumes, was related to the ROS formation. It is inferred that women exposed to emitted fumes from cooking oil were at higher risk of contracting lung cancer.     

Anti-skin cancer properties of phenolic-rich extract from the pericarp of mangosteen (Garcinia mangostana Linn.)

Skin cancers are often resistant to conventional chemotherapy. This study examined the anti-skin cancer properties of crude ethanol extract of mangosteen pericarp (MPEE) on human squamous cell carcinoma A-431 and melanoma SK-MEL-28 lines. Significant dose-dependent reduction in% viability was observed for these cell lines, with less effect on human normal skin fibroblast CCD-1064Sk and keratinocyte HaCaT cell lines. Cell distribution in G₁ phase (93%) significantly increased after 10 μg/ml of MPEE versus untreated SK-MEL-28 cells (78%), which was associated with enhanced p21^WAF1 mRNA levels. In A-431 cells, 10 μg/ml MPEE significantly increased the sub G₁ peak (15%) with concomitant decrease in G₁ phase over untreated cells (2%). In A-431 cells, 10 μg/ml MPEE induced an 18% increase in early apoptosis versus untreated cells (2%). This was via caspase activation (15-, 3- and 4-fold increased caspse-3/7, 8, and 9 activities), and disruption of mitochondrial pathways (6-fold decreased mitochondrial membrane potential versus untreated cells). Real-time PCR revealed increased Bax/Bcl-2 ratio and cytochrome c release, and decreased Akt1. Apoptosis was significantly increased after MPEE treatment of SK-MEL-28 cells. Hence, MPEE showed strong anti-skin cancer effect on these two skin cancer cell lines, with potential as an anti-skin cancer agent.     

Ginsenoside Rg1 reduces toxicity of PM2.5 on human umbilical vein endothelial cells by upregulating intracellular antioxidative state

Ambient airborne particulate matter (PM) is an important environmental pollutant responsible for many human diseases. Oxidative stress is suggested to be involved in PM-induced cell injury. The present study is designed to study unsalutary effects of the organic extracts of PM with an aerodynamic diameter of less than 2.5 μm (PM_2.5) and protective effect of Ginsenoside Rg1 (Rg1) against PM_2.5 on human umbilical vein endothelial cells (HUVECs) in vitro. Cytotoxic effects of the organic extract PM_2.5 on HUVECs were measured by means of HUVEC cell viability and the generation of intracellular reactive oxygen species (ROS). Expression of heme oxygenase-1(HO-1) and Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Nrf2 cytoplasm–nucleus location were assayed. The present results showed that PM_2.5 (50–800 μg/ml) decreased HUVEC viability and increased intracellular generation of ROS and malondialdehyde (MDA) in a concentration dependent manner, but increased HO-1 expression without concentration dependence. Rg1 (10 and 40 μg/ml) diminished PM_2.5-induced HUVEC viability, decrease ROS and MDA generation, increased HO-1 and Nrf2 expression and promoted Nrf2 translocation to nucleus in a concentration dependent manner. These results suggested that organic extracts of PM_2.5 increase oxidative stress and decrease cell viability; Rg1 antagonize PM_2.5-induced excess oxidative stress; HO-1 expression increase and Nrf2 translocation to nucleus may be involved in the effects of both PM_2.5 and Rg1 on HUVECs.