Synthesis of biogenic silver nanoparticles using Althaea officinalis as reducing agent: evaluation of toxicity and ecotoxicity.

Silver nanoparticles (AgNPs) are known mainly because of their bactericidal properties. Among the different types of synthesis, there is the biogenic synthesis, which allows the synergy between the nanocomposites and substances from the organism employed for the synthesis. This study describes the synthesis of AgNPs using infusion of roots (AgNpR) and extract (AgNpE) of the plant Althaea officinalis. After the synthesis through reduction of silver nitrate with compounds of A. officinalis, physico-chemical analyzes were performed by UV-Vis spectroscopy, nanoparticles tracking analysis (NTA), dynamic light scattering (DLS) and scanning electron microscopy (SEM). Toxicity was evaluated through Allium cepa assay, comet test with cell lines, cell viability by mitochondrial activity and image cytometry and minimal inhibitory concentration on pathogenic microorganisms. Biochemical analyzes (CAT - catalase, GPx - glutathione peroxidase e GST - glutationa S-transferase) and genotoxicity evaluation in vivo on Zebrafish were also performed. AgNpE and AgNpR showed size of 157 ± 11 nm and 293 ± 12 nm, polydispersity of 0.47 ± 0.08 and 0.25 ± 0.01, and zeta potential of 20.4 ± 1.4 and 26.5 ± 1.2 mV, respectively. With regard to toxicity, the AgNpE were the most toxic when compared with AgNpR. Biochemical analyzes on fish showed increase of CAT activity in most of the organs, whereas GPx showed few changes and the activity of GST decreased. Also regarding to bactericidal activity, both nanoparticles were effective, however AgNpR showed greater activity. Althaea officinalis can be employed as reducing agent for the synthesis of silver nanoparticles, although it is necessary to consider its potential toxicity and ecotoxicity.

Highly efficient adenoviral transduction of pancreatic islets using a microfluidic device.

Tissues are challenging to genetically manipulate due to limited penetration of viral particles resulting in low transduction efficiency. We are particularly interested in expressing genetically-encoded sensors in ex vivo pancreatic islets to measure glucose-stimulated metabolism, however poor viral penetration biases these measurements to only a subset of cells at the periphery. To increase mass transfer of viral particles, we designed a microfluidic device that holds islets in parallel hydrodynamic traps connected by an expanding by-pass channel. We modeled viral particle flow into the tissue using fluorescently-labelled gold nanoparticles of varying sizes and showed a penetration threshold of only ∼5 nm. To increase this threshold, we used EDTA to transiently reduce cell-cell adhesion and expand intercellular space. Ultimately, a combination of media flow and ETDA treatment significantly increased adenoviral transduction to the core of the islet. As proof-of-principle, we used this protocol to transduce an ER-targeted redox sensitive sensor (eroGFP), and revealed significantly greater ER redox capacity at core islet cells. Overall, these data demonstrate a robust method to enhance transduction efficiency of islets, and potentially other tissues, by using a combination of microfluidic flow and transient tissue expansion.

Bio-screening of a few green seaweeds from India for their cytotoxic and antioxidant potential.

BACKGROUND: It has been evidenced in several epidemiological studies that seaweeds when consumed as diet protect against several chronic oxidative stress-related diseases. Seaweeds, raw, cooked, or dried, are used as food in many cultures, although not very popularly in India. Globally, several studies have indicated that seaweeds are a rich source of phenolic compounds and have antioxidant properties. In the present study, we screened methanolic extracts (MEs) of five species of green seaweeds commonly found in India for their cytotoxic activity by brine shrimp lethality assay and antioxidant properties using various in vitro assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, reducing power and metal ion chelating assays. RESULTS: A markedly variable, dose-dependent activity was observed in all the seaweed extracts relative to their total phenolic content. Statistical analysis indicated a significantly strong correlation between the DPPH radical scavenging activity and total phenolic content (R(2) = 0.88, P < 0.05) as well as reducing power and total phenolic content (R(2) = 0.99, P < 0.01) of the dry MEs. Also, a very poor correlation between total phenolic content and metal chelating activity (R(2) = 0.13, P > 0.05) was noted. None of the seaweed extracts were potently cytotoxic. CONCLUSION: The underlying results endorse seaweeds as a rich, novel source of antioxidant compounds needing systemic exploration.

Endoplasmic reticulum oxidoreductin-1-like ß (ERO1lß) regulates susceptibility to endoplasmic reticulum stress and is induced by insulin flux in ß-cells.

Hyperglycemia increases insulin flux through the endoplasmic reticulum (ER) of pancreatic ß-cells, and the unfolded protein response pathway is required to enhance insulin processing. Pancreatic and duodenal homeobox 1 (PDX1), a key pancreatic transcription factor, regulates insulin along with targets involved in insulin processing and secretion. Here we find that PDX1 is a direct transcriptional regulator of ER oxidoreductin-1-like ß (Ero1lß), which maintains the oxidative environment of the ER to facilitate disulfide bond formation. PDX1 deficiency reduced Ero1lß transcript levels in mouse islets and mouse insulinoma (MIN6) cells; moreover, PDX1 occupied the Ero1lß promoter in ß-cells. ERO1lß levels were induced by high glucose concentrations and by the reducing agent dithiothreitol, indicating potential roles in adaptation to increased oxidative protein folding load in the ß-cell ER. In MIN6 cells, small interfering RNA-mediated silencing of Ero1lß decreased insulin content and increased susceptibility to ER stress-induced apoptosis. These findings demonstrate roles for the PDX1 target ERO1lß in maintaining insulin content and regulating cell survival during ER stress.

Redox modulation at the peripheral site alters nociceptive transmission in vivo.

1. The aim of the present study was to investigate the role of redox modulation during the peripheral nociceptive transmission in vivo. The nociceptive response was evaluated by the amount of time that mice spent licking the footpad injected with glutamate (20 micromol/paw). Thiol groups in footpad tissue were quantified using a colourimetric reaction with 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). 2. When coadministered with glutamate, the thiol alkylating agent iodoacetate (200 nmol/paw) caused significant antinociception in footpad tissue, in parallel with a decrease in free thiol groups. Treatment with the reducing agent dithiothreitol (200 nmol/paw) 5 min before glutamate and iodoacetate prevented the antinociception and thiol loss caused by iodoacetate. Injection of 100 nmol/paw ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), an in vitro redox modulator of the N-methyl-d-aspartate (NMDA) receptor, also prevented iodoacetate-induced antinociception. However, ebselen did not prevent thiol loss in the footpad. Dithiothreitol and ebselen had a synergic nociceptive effect with glutamate. 3. Alone, ebselen (100 nmol/paw) exhibited a pronociceptive effect. The nociception induced by ebselen was blocked by glutathione depletion induced by buthionine-sulphoximine (BSO; 2.5 micromol/paw). In addition, ebselen-induced nociception was prevented by 75 +/- 2% following injection of 5 nmol/paw MK-801 (an NMDA receptor antagonist). The nitric oxide synthase inhibitor N(G)-nitro-l-arginine (250 nmol/paw) had no effect on the nociception produced by ebselen. 4. In conclusion, the present paper reports on the effect of redox modulation on the glutamatergic system during peripheral nociceptive transmission in vivo. Antinociception was directly correlated with the availability of thiol groups, whereas the pronociceptive response of the reducing agents likely occurs via positive modulation of the NMDA receptor.

Immobilization of simulated reducing agent at the surface of SiO2 fillers in dental composite resins.

To reduce the leachability of reducing agents from composite resins, immobilization of a simulated reducing agent at the surface of SiO2 fillers was examined. SiO2 plates were immersed in 2% 3-aminopropyltriethoxy silane/ethanol solution, and then immersed in dimethyl sulfoxide with 0.25 wt% 4-dimethyl amino benzoic acid (DMABA), 2.0 wt% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 0.5 wt% N-hydroxysuccinimide. Wide-scan spectrum of X-ray photoelectron spectroscopy did not detect carbon contamination. However, narrow scan detected an O=C-N peak at 399.8 eV, suggesting that DMABA could be immobilized on silane-coupled SiO2 plates. Further, surface plasmon resonance analysis indicated the adsorption of MMA at the surface of reducing agent-immobilized plate.

Development of gene vectors for pinpoint targeting to human hepatocytes by cationically modified polymer complexes.

We developed a vector that might enable gene therapy of metabolic liver disease or hepatoma. Here we demonstrate the use of cationically modified biocompatible phospholipid polymer conjugated with hepatitis B surface (HBs) antigen for the specific transfer of genes into human hepatocytes. Poly(2-methacryloyloxyethyl phosphorylcholine (MPC)- co-N,N-dimethylaminoethyl methacrylate (DMAEMA)-co- p-nitrophenylcarbonyloxyethyl methacrylate(NPMA))(polyMDN) was prepared as a frame of vector. The specific expression of sFlt-1 or GFP by polyMDN conjugated with HBs containing plasmid (plasmid/polyMDN-HBs), polyMDN containing plasmid (plasmid/polyMDN), plasmid only and PBS were assessed in tumor cells (HepG2 or WiDr) in vitro and in vivo. The histological findings, organ weight changes, and degree of liver dysfunction were examined in the mice administered by several reagents. The sFlt-1 and GFP expression was observed only in the HepG2 cells transfected with sFlt-1 or GFP/polyMDN-HBs. None of the side effects mentioned above was observed. In conclusion, these results suggest that polyMDN-HBs is a human hepatocyte-specific gene delivery vector that might not have serious side effects.

Apoptotic and necrotic influence of dental resin polymerization initiators in human gingival fibroblast cultures.

The aim of this study was to examine the apoptotic and necrotic influence of four dental resin polymerization initiators--namely benzoyl peroxide (BPO), camphorquinone (CQ), dimethylaminoethyl methacrylate (DMAEMA), and dimethyl-para-toluidine (DMPT)--on human gingival fibroblast (HGF) cells. To this end, the growth inhibition of HGF cells with 1 mM BPO, CQ, and DMAEMA, and 500 microM DMPT was evaluated using Cell Counting Kit-8. Then, cell cycle analysis by flow cytometry was used to assess propidium iodide-stained cells (distribution of cells in G0/G1, S, G2/M phases). All four dental resin polymerization initiators induced G0/G1 cell cycle arrest. As for the patterns of cell death (necrosis and/or apoptosis), they were analyzed using Annexin V-FITC/PI staining with flow cytometry. All four dental resin polymerization initiators most likely induced necrosis.

Cytoprotective effects of Lycium barbarum against reducing stress on endoplasmic reticulum.

Chinese medicinal herbs have been consumed for thousands of years for the purpose of healthy aging. Lycium barbarum is valued in Chinese culture for its benefits to anti-aging, vision, kidney and liver. Recent studies showed that extracts from L. barbarum possess biological activities including anti-aging, anti-tumor, immune-stimulatory and cytoprotection. Most of these studies emphasized that the protective function of L. barbarum is due to its anti-oxidative effects. We have previously demonstrated that extract from L. barbarum can protect neurons against beta-amyloid (Abeta) peptide-induced apoptosis. Since Abeta toxicity may be mediated via oxidative stress, it is still unclear whether the extract from L. barbarum is a simple anti-oxidant exhibiting cytoprotective effects. We hypothesized that extract from L. barbarum is not simply an anti-oxidant in order to function as a neuroprotective agent. The aim of this study is to investigate whether the extract from L. barbarum (LBG) protect neurons via mechanisms independent of anti-oxidative effects. Using a reducing agent, dithiothreitol (DTT), we found that LBG exhibits cytoprotective effects against reducing stress by lowering the DTT-induced LDH release and caspase-3 activity. DTT can trigger endoplasmic reticulum (ER) stress leading to PKR-like ER kinase (PERK) activation. We also showed that LBG attenuates DTT-induced PERK phosphorylation. The extract from L. barbarum is not simply an anti-oxidant; it can also exhibit cytoprotective effects against reducing stress by DTT.

Genotoxicity of dental resin polymerization initiators in vitro.

The polymerization initiators for resins cured using visible light usually consist of a photosensitizer, primarily camphorquinone (CQ), and a reducing agent, which is often a tertiary amine (DMPT, DMAEMA), while the initiator used for self-curing resins consists of benzoyl peroxide (BPO) and a tertiary amine (DMPT). The genotoxicities of camphorquinone (CQ), benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), 2-dimethylamino-ethyl-methacrylate (DMAEMA), and 1-allyl-2-thiourea (ATU) were examined using the bioluminescent bacterial genotoxicity test. 4-Nitroquinoline-N-oxide (4NQO) was prepared for comparison with these chemicals. Acetone solutions of the five polymerization initiators and 4NQO were prepared. Benzoyl peroxide (BPO), dimethyl-para-toluidine (DMPT), and 1-allyl-2-thiourea (ATU) showed significant genotoxic activity at 24 h in the bioluminescent bacterial genotoxicity test, at concentrations of approximately 5 microM, 4 mM, and 1 mM, respectively. 2-Dimethyloamino-ethyl-methacrylate (DMAEMA) did not have genotoxic activity and CQ had questionable genotoxic activity. In comparison, 4NQO had strong genotoxicity, at 4 microM, roughly the same as that of BPO. Therefore, BPO should be used carefully in clinical dentistry.

Comparative radical production and cytotoxicity induced by camphorquinone and 9-fluorenone against human pulp fibroblasts.

Camphorquinone (CQ) is widely used as a photo-initiator in dental materials; however, its cytotoxicity against human pulp fibroblasts (HPF) and particularly the effects of 2-dimethylaminoethyl methacrylate (DMA), a reducing agent and visible light (VL) irradiation on it remain unknown. So we investigated the cytotoxic and reactive oxygen species (ROS)-producing effects of CQ with or without DMA, in the presence or absence of VL on HPF cells. The free-radical production activity of CQ was measured by two different methods [using diphenylpicryl hydrazyl and galvinoxyl]. The phase-transition properties of dipalmitoylphosphatidyl choline (DPPC) liposomes, as a model for biomembranes, induced by CQ were investigated by differential scanning calorimetry. These findings were compared with those of 9-fluorenone (9F), an aromatic photo-initiator with long conjugated groups. Camphorquinone with VL irradiation increased the radical production, whereas 9F with VL irradiation increased ROS production, as well as effecting changes in the DPPC phase-transition properties. The cytotoxicity of CQ towards HPF cells was smaller than that of 9F despite greater radical production. The addition of DMA to the photosensitizer enhanced the free-radical production without increasing the ROS level or the cytotoxicity. Camphorquinone/DMA is a valuable combination for the polymerization of dental resins.

Final report on the safety assessment of sodium sulfite, potassium sulfite, ammonium sulfite, sodium bisulfite, ammonium bisulfite, sodium metabisulfite and potassium metabisulfite.

Sodium Sulfite, Ammonium Sulfite, Sodium Bisulfite, Potassium Bisulfite, Ammonium Bisulfite, Sodium Metabisulfite, and Potassium Metabisulfite are inorganic salts that function as reducing agents in cosmetic formulations. All except Sodium Metabisulfite also function as hair-waving/straightening agents. In addition, Sodium Sulfite, Potassium Sulfite, Sodium Bisulfite, and Sodium Metabisulfite function as antioxidants. Although Ammonium Sulfite is not in current use, the others are widely used in hair care products. Sulfites that enter mammals via ingestion, inhalation, or injection are metabolized by sulfite oxidase to sulfate. In oral-dose animal toxicity studies, hyperplastic changes in the gastric mucosa were the most common findings at high doses. Ammonium Sulfite aerosol had an acute LC(50) of >400 mg/m(3) in guinea pigs. A single exposure to low concentrations of a Sodium Sulfite fine aerosol produced dose-related changes in the lung capacity parameters of guinea pigs. A 3-day exposure of rats to a Sodium Sulfite fine aerosol produced mild pulmonary edema and irritation of the tracheal epithelium. Severe epithelial changes were observed in dogs exposed for 290 days to 1 mg/m(3) of a Sodium Metabisulfite fine aerosol. These fine aerosols contained fine respirable particle sizes that are not found in cosmetic aerosols or pump sprays. None of the cosmetic product types, however, in which these ingredients are used are aerosolized. Sodium Bisulfite (tested at 38%) and Sodium Metabisulfite (undiluted) were not irritants to rabbits following occlusive exposures. Sodium Metabisulfite (tested at 50%) was irritating to guinea pigs following repeated exposure. In rats, Sodium Sulfite heptahydrate at large doses (up to 3.3 g/kg) produced fetal toxicity but not teratogenicity. Sodium Bisulfite, Sodium Metabisulfite, and Potassium Metabisulfite were not teratogenic for mice, rats, hamsters, or rabbits at doses up to 160 mg/kg. Generally, Sodium Sulfite, Sodium Metabisulfite, and Potassium Metabisulfite were negative in mutagenicity studies. Sodium Bisulfite produced both positive and negative results. Clinical oral and ocular-exposure studies reported no adverse effects. Sodium Sulfite was not irritating or sensitizing in clinical tests. These ingredients, however, may produce positive reactions in dermatologic patients under patch test. In evaluating the positive genotoxicity data found with Sodium Bisulfite, the equilibrium chemistry of sulfurous acid, sulfur dioxide, bisulfite, sulfite, and metabisulfite was considered. This information, however, suggests that some bisulfite may have been present in genotoxicity tests involving the other ingredients and vice versa. On that basis, the genotoxicity data did not give a clear, consistent picture. In cosmetics, however, the bisulfite form is used at very low concentrations (0.03% to 0.7%) in most products except wave sets. In wave sets, the pH ranges from 8 to 9 where the sulfite form would predominate. Skin penetration would be low due to the highly charged nature of these particles and any sulfite that did penetrate would be converted to sulfate by the enzyme sulfate oxidase. As used in cosmetics, therefore, these ingredients would not present a genotoxicity risk. The Cosmetic Ingredient Review Expert Panel concluded that Sodium Sulfite, Potassium Sulfite, Ammonium Sulfite, Sodium Bisulfite, Ammonium Bisulfite, Sodium Metabisulfite, and Potassium Metabisulfite are safe as used in cosmetic formulations.

Combination of dithiothreitol and detergent treatment of spermatozoa causes paternal chromosomal damage.

Treatment of spermatozoa with either the nonionic detergent Triton X-100 (TX) or dithiothreitol (DTT) has been suggested to confer enhanced success on intracytoplasmic sperm injection (ICSI) in mice and humans. Here, we attempted to use both reagents together, to our knowledge for the first time, and found that this caused severe chromosomal breaks in paternal pronuclei. We documented this effect further by treating mouse spermatozoa with several combinations of DTT with and without detergent. Spermatozoa were treated with vigorous pipetting to induce membrane disruption or with TX or the ionic detergent mixed alkyltrimethylammonium bromide (ATAB). Swim-up spermatozoa were used as controls. In each treatment, two samples were tested, with or without the addition of DTT during the treatment procedure. In all samples with DTT, protamine reduction was confirmed by the decondensation assay. Sperm nuclei obtained after different treatments were injected into oocytes for cytogenetic analysis, and paternal and maternal chromosomes of the zygote were visualized and examined. We found that the numbers of normal paternal karyoplates resulting from ICSI with spermatozoa treated with either DTT (87%, 153/176), TX (79%, 112/142), or ATAB (85%, 99/116) alone were similar to swim-up controls (92%, 103/112). However, only 22% (23/103) and 40% (59/149) of examined metaphases were scored as normal in TX + DTT or ATAB + DTT treatments, respectively. Spermatozoa in which the membranes were disrupted by vigorous pipetting in the presence of DTT had a slightly reduced frequency of normal chromosomes (61%, 64/104), whereas those without DTT were normal (79%, 125/159). However, this difference was not statistically significant. When spermatozoa were treated with TX + DTT in the presence of EGTA or a mixture of EGTA and EDTA, the frequency of normal chromosomes was 39% (45/114) and 47% (38/81), respectively, suggesting that endogenous sperm nucleases may play a role in chromosomal damage. Our results indicate that simultaneous treatment of spermatozoa with detergent and DTT induces extensive chromosomal breakage and, therefore, should not be attempted in ICSI.

In vitro evaluation of 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-trifluoromethylquinoline hydrochloride (NLTQ-1), a new bioreductive agent as a hypoxia marker by 19F-magnetic resonance spectroscopy (19F-MRS).

19F-labeled bioreductive drugs bound to hypoxic cells in tumors could be detected by nuclear magnetic resonance, provided that they do not lose 19F during their metabolism. NLTQ-1, a 2-nitroimidazole-linked 7-trifluoromethylquinoline, has been synthesized to furnish this aim. NLTQ-1 demonstrated hypoxic selectivities of 7-10 in various cell-lines, in vitro. Uptake studies in V79 cells showed a 5 to 6 fold greater intracellular than extracellular concentration at a range of 100-300 microM input concentrations. A strong sharp peak, which was identified as the parent compound, was observed in the 19F-NMR spectrum of 90% MeCN extracts of V79 cells aerobically exposed to NLTQ-1, indicating that NLTQ-1 was not metabolized under aerobic conditions. Similarly, 19F NMR efflux studies in intact cells showed that the NLTQ-1 was bound to the cells predominantly under hypoxic conditions. 19F-NMR spectra of intact cells, exposed under hypoxic conditions to NLTQ-1, and of their lysates, after precipitation of various cellular components, indicated that possible covalent binding of NLTQ-1 had occurred with macromolecules such as proteins and nucleic acids. Therefore, NLTQ-1 might be suitable as a 19F-MRS/MRI hypoxia probe, although further in vivo work is necessary to verify this matter.