Hydrofluoric acid treatment of titanium surfaces enhances the proliferation of human gingival fibroblasts.

The attachment of implants relies on bone and soft tissue biocompatibility. The aim of this article is to investigate the effect of fluoride-modified metallic titanium (Ti) surfaces (Ti-F) on proliferation and differentiation of human gingival fibroblasts. Human gingival fibroblast cells were exposed to hydrofluoric acid-modified Ti coins (Ti-F) for 1, 3, 7, 14 and 21 days, and untreated coins were used as controls. A five- to six-fold increase in the proliferation of human gingival fibroblasts on Ti-F compared to Ti surfaces was observed. Enhanced gene expression of interleukin-6 and osteoprotegerin was found at 7 days. Increased levels of sclerostin, interleukin-6 and osteoprotegerin in the media from human gingival fibroblasts cultured on Ti-F coins were found compared to controls. Our results confirm that hydrofluoric acid-modified surface may indirectly enhance the firm attachment of implant surface to junction epithelium, soft tissue epithelium, which would give protection for underlying osseous structures making osseointegration of the dental implant possible.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

Effect of silver nanoparticles on mitogen-activated protein kinases activation: role of reactive oxygen species and implication in DNA damage.

Large quantities of engineered nanoparticles (NP), such as nanosilver (AgNP), have been widely applied, leading to an increased exposure and potential health concerns. Herein, we have examined the ability of AgNP to induce reactive oxygen species (ROS), their role in genotoxic effects and the involvement of mitogen-activated protein kinases (MAPK). AgNP exposure induced ROS production in human epithelial embryonic cells which could be decreased by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation, induced by AgNP, was an early response but not sustained in time. Furthermore, JNK and ERK activation could be inhibited by both DPI and a free radicals scavenger N-acetyl cysteine. We also investigated the role of MAPK in the DNA damage. Using a modified comet assay for the specific detection of hOGG1 sensitive sites, we showed that AgNP induced DNA oxidation after 30-min treatment, whereas no response was observed after 2h. In conclusion, AgNP seem to induce DNA damage via a mechanism involving ROS formation. The oxidative DNA damage observed was transient, likely due to DNA repair; furthermore, higher damage was achieved upon inhibition of ERK activation by pre-treatment with U0126, suggesting a role for ERK in DNA damage repair. Activation of different MAPK might play an important role in the NP toxicity outcomes; understanding this process may be helpful for the identification of NP toxicity.

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles.

Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.

Silver nanoparticles induce premutagenic DNA oxidation that can be prevented by phytochemicals from Gentiana asclepiadea.

Among nanomaterials, silver nanoparticles (AgNPs) have the broadest and most commercial applications due to their antibacterial properties, highlighting the need for exploring their potential toxicity and underlying mechanisms of action. Our main aim was to investigate whether AgNPs exert toxicity by inducing oxidative damage to DNA in human kidney HEK 293 cells. In addition, we tested whether this damage could be counteracted by plant extracts containing phytochemicals such as swertiamarin, mangiferin and homoorientin with high antioxidant abilities. We show that AgNPs (20 nm) are taken up by cells and localised in vacuoles and cytoplasm. Exposure to 1, 25 or 100 µg/ml AgNPs leads to a significant dose-dependent increase in oxidised DNA base lesions (8-oxo-7,8-dihydroguanine or 8-oxoG) detected by the comet assay after incubation of nucleoids with 8-oxoG DNA glycosylase. Oxidised DNA base lesions and strand breaks caused by AgNPs were diminished by aqueous and methanolic extracts from both haulm and flower of Gentiana asclepiadea.

Gentiana asclepiadea protects human cells against oxidation DNA lesions.

The objectives of this study were to examine whether the methanolic and aqueous extracts from the haulm and flower of Gentiana asclepiadea exhibited free radical scavenging and protective (antigenotoxic) effect against DNA oxidation induced by H(2)O(2) in human lymphocytes and human embryonic kidney cells (HEK 293). All four extracts exhibited high scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at concentrations 2.5 and 25 mg ml(-1). The level of DNA damage was measured using the alkaline version of single-cell gel electrophoresis (comet assay). Challenge with H(2)O(2) shows that the pre-treatment of the cells with non-genotoxic doses of Gentiana extracts protected human DNA-either eliminated or significantly reduced H(2)O(2) induced DNA damage. The genotoxic activity of H(2)O(2) was most effectively decreased after 30 min of pre-incubation with 0.05 mg ml(-1) (range, 93.5%-96.3% of reduction in lymphocytes) and 0.25 mg ml(-1) (range, 59.5%-71.4% and 52.7%-66.4% of reduction in lymphocytes and HEK 293 cells, respectively) of G. asclepiadea extracts. These results suggest that the tested G. asclepiadea extracts could be considered as an effective natural antioxidant source.

Exacerbation of tobacco smoke mediated apoptosis by resveratrol: an unexpected consequence of its antioxidant action.

Resveratrol, a polyphenolic compound rich in grapes and red wine, has been reported to protect cells against oxidative damage and cell death by increasing cellular antioxidant/detoxification capacity. Cigarette smoking is a major risk factor for respiratory diseases and oxidative damage is implicated in its pathogenesis. Here we investigated the enhancement of antioxidant capacity by resveratrol and its potential protection against cell death caused by cigarette smoke in human bronchial epithelial cells (HBE1). At concentrations that did not affect cell growth, resveratrol activated Nrf2 signaling and increased the expression of NAD(P)H:quinone reductase-1, heme oxygenase-1, and the catalytic subunit of glutamate cysteine ligase. Surprisingly, instead of protecting against cell death, resveratrol significantly enhanced cigarette smoke extract-induced apoptosis. To define the underlying mechanism, the effect of resveratrol on caspase activity was examined and it was found that resveratrol significantly enhanced cigarette smoke-stimulated caspase activity. In conclusion, results from this study suggest that although resveratrol increased antioxidant and detoxification capacity, it increased rather than protected against cigarette smoke-induced apoptosis.

Cytotoxic and genotoxic effect of methanolic flower extract from Gentiana asclepiadea on COS 1 cells.

OBJECTIVE: The purpose of this study was to assess whether a methanol extract isolated from the flower of Gentiana asclepiadea had potential cytotoxic or genotoxic effect on COS 1 (monkey kidney) cell line. Five various concentrations of the extract were investigated for cytotoxicity and genotoxicity and to determine non-cytotoxic and non-genotoxic concentrations suitable for utilization in pharmacology and medicine. METHODS: Cytotoxicity was determined using the proliferation (growth activity) and the plating efficiency (colony forming ability) assays after 24 hour incubation of COS 1 cells with different concentrations of methanolic flower extract from G. asclepiadea. To assess potential genotoxicity, the comet assay or SCGE (Single-Cell Gel Electrophoresis) was used. RESULTS: We found that only the highest (5 and 25 mg/ml) concentrations of the extract revealed cytotoxic and genotoxic effect. We have also determined concentrations that stimulated cell growth (0.25 mg/ml) and colony forming ability (0.25-2.5 mg/ml) and did not exhibit genotoxic effect (0.25-2.5 mg/ml). CONCLUSIONS: We found out that extract of G. asclepiadea was neither cytotoxic nor genotoxic in a wide range of concentrations (0.25-2.5 mg/ml) and thus can be used to further investigate potential beneficial usage in pharmacology and medicine.

Multidrug-resistant protein-3 gene regulation by the transcription factor Nrf2 in human bronchial epithelial and non-small-cell lung carcinoma.

Multidrug-resistant proteins (MRPs) are members of the ATP-binding cassette superfamily that facilitate detoxification by transporting toxic compounds, including chemotherapeutic drugs, out of cells. Chemotherapy, radiation, and other xenobiotic stresses have been shown to increase levels of select MRPs, although the underlying mechanism remains largely unknown. Additionally, MRP3 is suspected of playing a role in the drug resistance of non-small-cell lung carcinoma (NSCLC). Analysis of the MRP3 promoter revealed the presence of multiple putative electrophile-responsive elements (EpREs), sequences that suggest possible regulation of this gene by Nrf2, the key transcription factor that binds to EpRE. The goal of this investigation was to determine whether MRP3 induction was dependent upon the transcription factor Nrf2. Keap1, a key regulator of Nrf2, sequesters Nrf2 in the cytoplasm, preventing entry into the nucleus. The electrophilic lipid peroxidation product 4-hydroxy-2-nonenal (HNE) has been shown to modify Keap1, allowing Nrf2 to enter the nucleus. We found that HNE up-regulated MRP3 mRNA and protein levels in cell lines with wild-type Keap1 (the human bronchial epithelial cell line HBE1 and the NSCLC cell line H358), but not in the Keap1-mutant NSCLC cell lines (A549 and H460). Cell lines with mutant Keap1 had constitutively higher MRP3 that was not increased by HNE treatment. In HBE1 cells, silencing of Nrf2 with siRNA inhibited induction of MRP3 by HNE. Finally, we found that silencing Nrf2 also increased the toxicity of cisplatin in H358 cells. The combined results therefore support the hypothesis that MRP3 induction by HNE involves Nrf2 activation.

Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells.

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.

Glutathione: overview of its protective roles, measurement, and biosynthesis.

This review is the introduction to a special issue concerning, glutathione (GSH), the most abundant low molecular weight thiol compound synthesized in cells. GSH plays critical roles in protecting cells from oxidative damage and the toxicity of xenobiotic electrophiles, and maintaining redox homeostasis. Here, the functions and GSH and the sources of oxidants and electrophiles, the elimination of oxidants by reduction and electrophiles by conjugation with GSH are briefly described. Methods of assessing GSH status in the cells are also described. GSH synthesis and its regulation are addressed along with therapeutic approaches for manipulating GSH content that have been proposed. The purpose here is to provide a brief overview of some of the important aspects of glutathione metabolism as part of this special issue that will provide a more comprehensive review of the state of knowledge regarding this essential molecule.

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal.

During the past several years, major advances have been made in understanding how reactive oxygen species (ROS) and nitrogen species (RNS) participate in signal transduction. Identification of the specific targets and the chemical reactions involved still remains to be resolved with many of the signaling pathways in which the involvement of reactive species has been determined. Our understanding is that ROS and RNS have second messenger roles. While cysteine residues in the thiolate (ionized) form found in several classes of signaling proteins can be specific targets for reaction with H(2)O(2) and RNS, better understanding of the chemistry, particularly kinetics, suggests that for many signaling events in which ROS and RNS participate, enzymatic catalysis is more likely to be involved than non-enzymatic reaction. Due to increased interest in how oxidation products, particularly lipid peroxidation products, also are involved with signaling, a review of signaling by 4-hydroxy-2-nonenal (HNE) is included. This article focuses on the chemistry of signaling by ROS, RNS, and HNE and will describe reactions with selected target proteins as representatives of the mechanisms rather attempt to comprehensively review the many signaling pathways in which the reactive species are involved.

SHP-1 inhibition by 4-hydroxynonenal activates Jun N-terminal kinase and glutamate cysteine ligase.

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, is toxic at high concentrations, but at near-physiological concentrations it induces detoxifying enzymes. Previous data established that in human bronchial epithelial (HBE1) cells, both genes for glutamate cysteine ligase (GCL) are induced by HNE through the c-Jun N-terminal kinase (JNK) pathway. The protein-tyrosine phosphatase SH2 domain containing phosphatase-1 (SHP-1) is thought to play a role as a negative regulator of cell signaling, and has been implicated as such in the JNK pathway. In the present study, SHP-1 was demonstrated to contribute to HNE-induced-gclc expression via regulation of the JNK pathway in HBE1 cells. Treatment of HBE1 cells with HNE induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4), JNK, and c-Jun. HNE was able to inhibit protein tyrosine phosphatase activity of SHP-1 through increased degradation of the protein. Furthermore, transfection with small interference RNA SHP-1 showed an enhancement of JNK and c-Jun phosphorylation, but not of MKK4, leading to increased gclc expression. These results demonstrate that SHP-1 plays a role as a negative regulator of the JNK pathway and that HNE activated the JNK pathway by inhibiting SHP-1. Thus, SHP-1 acts as a sensor for HNE and is responsible for an important adaptive response to oxidative stress.

ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages.

Secretion of the proinflammatory cytokines, interleukin (IL)-1beta and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), whose inactivation would shift the equilibrium in favor of PI3K. ATP-dependent ROS production and PI3K activation also stimulate transcription of genes required for an oxidative stress response. In parallel, ATP-mediated ROS-dependent PI3K is required for activation of caspase-1 and secretion of IL-1beta and IL-18. Thus, an increase in ROS levels in ATP-treated macrophages results in activation of a single pathway that promotes both adaptation to subsequent exposure to oxidants or inflammation, and processing and secretion of proinflammatory cytokines.

The ADP-stimulated NADPH oxidase activates the ASK-1/MKK4/JNK pathway in alveolar macrophages.

The role of H2O2 as a second messenger in signal transduction pathways is well established. We show here that the NADPH oxidase-dependent production of O2*(-) and H2O2 or respiratory burst in alveolar macrophages (AM) (NR8383 cells) is required for ADP-stimulated c-Jun phosphorylation and the activation of JNK1/2, MKK4 (but not MKK7) and apoptosis signal-regulating kinase-1 (ASK1). ASK1 binds only to the reduced form of thioredoxin (Trx). ADP induced the dissociation of ASK1/Trx complex and thus resulted in ASK1 activation, as assessed by phosphorylation at Thr845, which was enhanced after treatment with aurothioglucose (ATG), an inhibitor of Trx reductase. While dissociation of the complex implies Trx oxidation, protein electrophoretic mobility shift assay detected oxidation of Trx only after bolus H2O2 but not after ADP stimulation. These results demonstrate that the ADP-stimulated respiratory burst activated the ASK1-MKK4-JNK1/c-Jun signaling pathway in AM and suggest that transient and localized oxidation of Trx by the NADPH oxidase-mediated generation of H2O2 may play a critical role in ASK1 activation and the inflammatory response.

Stimulation of the alveolar macrophage respiratory burst by ADP causes selective glutathionylation of protein tyrosine phosphatase 1B.

H(2)O(2) produced by stimulation of the macrophage NADPH oxidase is involved both in bacterial killing and as a second messenger in these cells. Protein tyrosine phosphatases (PTPs) are targets for H(2)O(2) signaling through oxidation of their catalytic cysteine, resulting in inhibition of their activity. Here, we show that, in the rat alveolar macrophage NR8383 cell line, H(2)O(2) produced through the ADP-stimulated respiratory burst induces the formation of a disulfide bond between PTP1B and GSH that was detectable with an antibody to glutathione-protein complexes and was reversed by DTT addition. PTP1B glutathionylation was dependent on H(2)O(2) as the presence of catalase at the time of ADP stimulation inhibited the formation of the conjugate. Interestingly, other PTPs, i.e., SHP-1 and SHP-2, did not undergo glutathionylation in response to ADP stimulation of the respiratory burst, although glutathionylation of these proteins could be shown by reaction with 25 mM glutathione disulfide in vitro. While previous studies have suggested the reversible oxidation of PTP1B during signaling or showed PTP1B glutathionylation in vitro, the present study directly demonstrates that physiological stimulation of H(2)O(2) production results in PTP1B glutathionylation in intact cells, which may affect downstream signaling.

Characterization of the hsp70 response in lymphoblasts from aged and centenarian subjects and differential effects of in vitro zinc supplementation.

Human centenarians attract increasing interest as they hold some still undefined molecular mechanisms resulting in the achievement of exceptional old age. Recent data suggest the ability of centenarians to efficiently counter the increased cellular stress normally associated with ageing. The ubiquitous heat shock (HS) protein HSP70, expressed under the control of the heat shock transcription factor 1 (HSF-1), is recognized as one of the main chaperones associated with cell protection against stresses. In fact, HSP70 protein induction by heat, a classic well characterized cellular stress, was recently reported to be reduced in cells of most aged humans but not in centenarians. In order to investigate the molecular basis of this feature, we analyzed in vitro the time course expression of the hsp70 gene and the activation of HSF-1 in heat treated Epstein Barr virus transformed B-lymphocytes of centenarians. Our study demonstrates that lymphoblasts from centenarians maintain the transcriptional response of hsp70 gene to heat stress similar to young subjects. Such normal induction of hsp70 is associated to higher binding activity of HSF-1 that compensates an age-dependent delay in HSF-1 phosphorylation. Moreover, in vitro zinc supplementation had an age-dependent effect on hsp70 expression, indicating a role for this nutritionally important molecule and suggesting its involvement in cellular stress responses.

Nutritional flavonoids modulate estrogen receptor alpha signaling.

Estrogen receptor alpha (ERalpha) mediates 17beta-estradiol (E2) actions through the transcription of E2-sensitive target genes. In addition, rapid non-genomic signaling (e.g., MAPK/ERK) occurs. It is now well accepted that these rapid membrane-initiated responses account for E2-related cancer. Beside many beneficial effects on human health, nutritional flavonoids exert protective and anticarcinogenic effects on E2-related cancer. The mechanism underlying these effects seems to be related to flavonoids antioxidant properties and/or to their ability to alter signal transduction protein kinases. In addition, an antiestrogenic activity has been proposed but not yet defined. However, the identification and characterization of the responsible mechanisms for flavonoid antitumoral effects is poorly understood. Here, we investigated the possibility that the antimitogenic effects of flavonoids are transduced by modulating ERalpha-mediated rapid signaling. The ability of two flavonoids, the flavanone naringenin and the flavanol quercetin, with respect of E2, to induce ERalpha activities has been studied in the human cervix epitheloid carcinoma cell line (HeLa) devoid of any estrogen receptors and rendered E2-sensitive by transient transfection with a human ERalpha expression vector. Our results indicate that flavonoids act as E2 mimetic on ERalpha transcriptional activity, whereas they impair the activation of rapid signaling pathways committed to E2-induced proliferation. The resulting decoupling of ERalpha signal transduction could be proposed as a new mechanism in the protective effects of flavonoids against E2-related cancer.