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Carbon nanodots modified with Neutral Red covalently inserted in the nanostructure (NR-CDs) have been prepared by a simple synthesis method based on microwave irradiation under controlled temperature and pressure. The synthetized NR-CDs have been characterized by different techniques, demonstrating the covalent bonding of Neutral Red molecules to the carbon dots nanostructure. Fluorescence activity of the prepare NR-CDs has been explored showing different interaction pathways with singled and doubled stranded DNA. These studies have been successfully applied to develop a new fluorescence DNA hybridization assay to the detection of a specific DNA sequence of Escherichia coli bacteria. Graphical abstract: [Figure not available: see fulltext.], Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work has been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (CTQ2017- 84309-C2-1-R; RED2018-102412-T) and Comunidad Autónoma de Madrid (SI3/PJI/2021–00341, P2018/NMT-4349 TRANSNANOAVANSENS Program)

Neutral Red (NR), a eurhodin dye, is often used for staining living cells, but we demonstrated for the first time that NR can also serve as a CO2 sensor, because of NR’s unique optical properties, which change with dissolved carbon dioxide (dCO2) concentrations. In the present study, optical sensitivity of NR was quantified as a function of changes in absorption and emission spectra to dCO2 in a pH 7.3 buffer medium at eight dCO2 concentrations. NR exhibited a response time of two minutes for equilibration in pure N2 to 100% CO2 with an ~200% percent change (%∆) in emission intensity and &gt, 400%∆ in absorbance, with full reversibility. Important to its application to biological systems, NR exhibited zero sensitivity to dissolved oxygen, which has routinely caused interference for CO2 measurements. NR exhibited pH sensitive emission and excitation energies with dual excitation wavelengths at 455 nm and 540 nm, and a single emission at 640 nm. The CO2 sensing properties of NR were benchmarked by a comparison to pyranine (8-hydroxypyrene-1, 3,6-trisulfonic acid trisodium salt) (HPTS). Future studies will evaluate the feasibility of NR as an intracellular in vivo pCO2 sensor in aquatic organisms critically impacted by increasing global CO2 levels.

Voltammetric DNA sensor has been proposed on the platform of glassy carbon electrode covered with carbon black with adsorbed pillar[5]arene molecules. Electropolymerization of Neutral Red performed in the presence of native or oxidatively damaged DNA resulted in formation of hybrid material which activity depended on the DNA conditions. The assembling of the surface layer was confirmed by scanning electron microscopy and electrochemical impedance spectroscopy. The influence of DNA and pillar[5]arene on redox activity of polymeric dye was investigated and a significant increase of the peak currents was found for DNA damaged by reactive oxygen species generated by Cu2+/H2O2 mixture. Pillar[5]arene improves the electron exchange conditions and increases the response and its reproducibility. The applicability of the DNA sensor developed was shown on the example of ascorbic acid as antioxidant. It decreases the current in the concentration range from 1.0 &mu, M to 1.0 mM. The possibility to detect antioxidant activity was qualitatively confirmed by testing tera infusion. The DNA sensor developed can find application in testing of carcinogenic species and searching for new antitumor drugs.

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Dredged material can cause several negative impacts to the ecosystems. Its management is a key issue for the protection of the aquatic environments. Sediment quality guidelines alone are not able to estimate the potential adverse effects that contaminants bound to sediment can cause. The in situ approach using caged bioindicator species has been chosen lately as a new sensitive tool for the assessment of dredged material. In a tier testing approach, Neutral Red Retention Assay (NRRA) has been applied as a screening tool in order to detect adverse changes in health status associated to contaminated environments. Nevertheless, to the authors' knowledge, little is known about the application and validation of NRRA in sediment bioindicator species and under field conditions. Caged Ruditapes philippinarum and Carcinus maenas were exposed during 28 days to 3 potentially contaminated sites in the Algeciras Bay (SW Spain) and to a reference site in the Cadiz Bay (SW Spain). Health status of exposed organisms was measured through NRRA in haemolymph samples. No significant differences of Neutral Red Retention Time (NRRT) were measured neither in clams nor in crabs over time in the organisms from control site (70 ± 10min). However significant differences (p < 0.05) were found at the end of the exposure period in clams and crabs exposed to contaminated sediments from Algeciras Bay, showing a decrease of NRRTs from 30 to 70% compared to the control organisms. The results obtained confirmed the use of NRRA as a suitable and sensitive method to be used in the assessment of sediment quality.

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes . A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.

In a new, simple, and fast flow-cytometric method for the simultaneous measurement of phagocytic activity of human neutrophils and monocytes in whole blood, the fluorescence capability of the well-known vital stain, neutral red was used. The incubation of 0.5 ml heparinized human blood with the phagocytic stimulus of zymosan dose- and time-dependently increased the percentage and the red fluorescence intensity of both neutrophils (4.3 +/- 1.2 times) and monocytes (2.7 +/- 0.7 times) measured cytofluorimetrically. Decreased uptake of neutral red was observed in a patient with phagocytic disorder, based upon impaired engulfment of particles and production of reactive oxygen species. In a patient with chronic granulomatosis, however, no decrease of neutral red uptake was measured. Platelet activating factor and phorbol myristate acetate were also able to increase the uptake of neutral red by both monocytes and neutrophils, but to a lesser extent than zymosan. The advantage of this method is the possibility for the simultaneous measurement of phagocytic activities of monocytes and neutrophils stimulated by either particles or soluble activators. This method is suitable for the selective measurement of activation processes not related to the production of free radicals in the phagocytes.

When certain photosensitizing compounds are added to a variety of biological systems, subsequent exposure to visible light produces structural and functional changes in the biological system. These changes constitute a photodynamic effect. After cells in tissue culture have been subjected to the photodynamic action of compounds, cytotoxic alterations such as nuclear fragmentation and blister cytolysis occur (1, 2).

Background Betulin, a natural pentacyclic triterpene with the lupane structure that is present in significant amounts in the outer bark of birch, is known for its broad array of biological and pharmacological properties. Betulin has attracted attention as a potential, natural-origin antimicrobial substance. The literature describes it as selectively toxic to neoplastic cells but safe for normal cells. The research aim was to evaluate the basal cytotoxicity of betulin towards fish (BF-2) and murine (NIH/3T3) fibroblasts. We used four colorimetric tests that provide a preliminary evaluation of possible mechanisms of the cytotoxicity of a compound to assess the degree of the toxicity of betulin after 24, 48 and 72 h of incubation with cells: the MTT assay (mitochondrial activity assessment), the NRU assay (lysosomal membrane integrity assessment), the LDH assay (cellular membrane integrity assessment) and the SRB assay (total cellular protein content determination). Results The results revealed an exceptionally high sensitivity of mitochondria to the effect of betulin, with the other endpoints being less sensitive. Although murine fibroblasts were more vulnerable to the toxic effect of betulin than fish fibroblasts, the betulin CC50 values for both cell lines were comparable with analogous IC50 values determined by other researchers in studies involving cancerous cells. Conclusions The results indicate the need to verify the claim about the selective toxicity of betulin towards malignant cells and to conduct safety/toxicity tests before any potential therapeutic use of betulin in veterinary medicine.

Confirmation of the biological effectiveness of new ophthalmic preparations introduced in the market is an important element in maintaining the safety of using this type of medications. This study aimed to investigate the activity of Ozodrop® on human corneal and conjunctival epithelial cells, as well as its antibacterial and antifungal activity. Cytotoxicity analyses of ocular surface epithelial cells were performed in vitro by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Neutral Red uptake assays. The level of nitric oxide released by the cells was assessed by the Griess method. The reduction of the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical by the tested formulation was analyzed. Microbiological tests were also performed. It was found that the Ozodrop® preparation exhibited biological activity, but was less active than the reference antibiotics and the anti-yeast agent. The cytotoxic activity of the Ozodrop® formulation was dependent on the time of cell exposure to it. No toxic effect was observed in the short-term, for up to 3 h. It appeared after 24 h of exposure of the cells to the preparation. The drops showed antioxidant activity in the specified concentration range. They also stimulated the release of nitric oxide, mainly by corneal epithelial cells. The Ozodrop® formulation exhibits biological activity that can be considered useful in the treatment of infections in the front part of the eye.

Mushrooms produce a variety of bioactive antioxidant secondary metabolites including ectins, polysaccharides, pigments, phenolic compounds, sterols and terpenes. Extracellular and intracellular compounds produced by submerged liquid fermentation are important industrially and economically. Phanerochaete chrysosporium (PC) is the model white-rot fungus that easy cultivation on lignocellulose-containing substrates. PC can be used as a bioprotein source. Aims of this study were to determine the in vitro antioxidant, cytotoxic and genotoxic effects of hot water extract obtained from PC on human peripheral blood monunuclear cells (hPBMCs). Cytotoxicity was determined by lactate dehydrogenase (LDH) leakeage assay and neutral red (NR). Total antioxidant capacity (TAC) and total oxidant status (TOS) were detected to determine the oxidative damage. Genotoxicity was characterized by micronuclei and chromosome aberrations assays for specify DNA damage. PC (5-75 µg/ml) significantly increased antioxidant capacity and these doses did not cause any significant alterations to cytotoxicity on hPBMCs. The elavated doses of PC (5-250 µg/ml) did not cause increase in genotoxic. Whereas, 250 and 500 µg/ml doses of PC statistically increased TOS levels, NR uptake, LDH release, CA/cell frequency and MN formation however decreased TAC levels. This study is the first time on cytotoxicity, genotoxicity and oxidative damage of PC on hPBMCs. In conclusion, the consumption of PC can be safe for humans, but it has also exposure period and dose-dependent effects on inducing oxidative damage and toxicity on hPBMCs., Mushroomsproduce a variety of bioactive antioxidant secondary metabolites including ectins,polysaccharides, pigments, phenolic compounds, sterols and terpenes. Extracellularand intracellular compounds produced by submerged liquid fermentation are importantindustrially and economically. Phanerochaetechrysosporium (PC) is the model white-rotfungus that easy cultivation on lignocellulose-containing substrates. PCcan be used as a bioprotein source. Aims of this study were to determine thein vitro antioxidant, cytotoxic and genotoxiceffects of hot water extract obtained from PC on human peripheral blood monunuclearcells (hPBMCs). Cytotoxicity was determined by lactate dehydrogenase (LDH) leakeageassay and neutral red (NR). Total antioxidant capacity (TAC) and total oxidant status(TOS) were detected to determine the oxidative damage. Genotoxicity was characterizedby micronuclei and chromosome aberrations assays for specify DNA damage. PC (5-75µg/ml) significantly increased antioxidantcapacity and these doses did not cause any significant alterations to cytotoxicityon hPBMCs. The elavated doses of PC (5-250 µg/ml) did not cause increase in genotoxic.Whereas, 250 and 500 µg/ml doses of PC statistically increased TOS levels, NR uptake,LDH release, CA/cell frequency and MN formation however decreased TAC levels. Thisstudy is the first time on cytotoxicity, genotoxicity and oxidative damage of PCon hPBMCs. In conclusion, the consumption ofPC can be safe for humans, but it has also exposure period and dose-dependenteffects on inducing oxidative damage and toxicity on hPBMCs.

Desert truffles have high nutritional value and grow wild in the Mediterranean basin and Western Asia. Although, many studies were performed to evaluate truffles nutritious values and phytochemical composition, studies are limited to evaluate their anticancer and/ or immunomodulatory effects. Our study was conducted to evaluate the anticancer and immunomodulatory effects of Terfezia boudieri (desert truffle). Different solvent extracts were prepared from the truffle and MTT assay was used to measure their anticancer activity against cancer cell lines (T47D, MCF-7, MDA-MB231, HCT-116, and Hela). Total phenolic content in each extract was determined by using Folin-Ciocalteu reagent and qualitative phytochemical screening was performed using standard methods. The degree of apoptosis induction (using caspase 3 assay) and vascular endothelial growth factor expression were detected using standard kits. Also, ELISA was used to measure levels of IFN-γ, IL-2, IL-4, and IL-10 secreted by splenocytes after treatment with the extracts. The effect of the extracts on splenocytes proliferation was measured using MTT assay. Macrophage function was evaluated using nitro blue tetrazolium assay and pinocytosis function was evaluated using neutral red method. Terpenoids, phytosterols, and carbohydrates were present in all the solvent extracts, while tannins, alkaloids and flavonoids were detected only in aqueous/methanol and aqueous extracts. The highest total phenolic content was observed in aqueous and aqueous methanol extracts. The growth of cancer cell lines was inhibited by T. boudieri extracts in a dose dependent manner. N-hexane extract was the most potent against most cell lines. Aqueous/methanol extract showed high apoptosis induction and angiogenesis suppression effects. An increase in TH1 cytokines (IFN-γ, IL-2) level and a decrease in TH2 cytokine (IL-4) level were evident after lymphocytes stimulation by aqueous/methanol, n-hexane and ethyl acetate extracts of T. boudieri. Ethyl acetate extract of T. boudieri were the most potent extracts to stimulate lymphocytes proliferation while all other extracts showed moderate stimulation. Aqueous/methanol extract was the most active extract to stimulate phagocytosis. Ethyl acetate extract was the most active extract to stimulate pinocytosis. The use of T. boudieri provides variable health benefits. N-hexane, ethyl acetate, and aqueous/methanol extracts exhibited anticancer activities and are potent stimulators of innate and acquired immunity. Further testing is needed to identify the biologically active compounds and detect them quantitatively using GC-MS analysis.

Abstract Filter-feeding organisms accumulate xenobiotics and other substances in their tissues. They can be useful as sentinel organisms in biomonitoring of the marine compartment. Bivalve cellular immunity is ensured by phagocytosis and cytotoxic reactions carried out by hemocytes in a network with humoral responses. These can be affected by chemical contaminants in water that can be immunosuppressors also at a low concentration increasing the sensibility to pathogens. This work is an attempt to individuate cellular markers for pollution detection, investigating the effect of methylmercury (CH3HgCl) at different concentrations on the activity and hemocyte morphology of the Mediterranean mussel, Mytilus galloprovincialis. We assessed the effect of three sub-lethal concentrations of the organometal on the cellular morphology, the efficacy of phagocytosis toward yeast cells, the alteration of the lysosomal membrane and the ability to release cytotoxic molecules. The results provide information on the alteration of hemocyte viability, modification of the morphological and cytoskeletal features and besides the cellular spreading, intrinsic ability of motile cells was used as a complementary investigation method. Exposure to the contaminant affected the percentage of phagocytosis and the phagocytosis index. Moreover, morphological and cytoskeleton alteration, caused by the pollutant, leads to reduced ability to incorporate the target and adhere to the substrate and the low ability of cells to retain neutral red could depend on the effects of methylmercury on membrane permeability. These results reinforce the use of the Mediterranean mussel as model for the evaluation of environmental quality in aquatic ecosystems integrating the novel information about hemocyte functions and morphology sensibility to organic mercury. Graphic abstract

Background Increasing evidences indicate that exposure to multi-walled carbon nanotubes (MWCNTs) could induce adverse vascular effects, but the role of length of MWCNTs in determining the toxic effects is less studied. This study investigated the adverse effects of two well-characterized MWCNTs to human umbilical vein endothelial cells (HUVECs). Methods The internalization and localization of MWCNTs in HUVECs were examined by using transmission electron microscopy (TEM). The cytotoxicity of MWCNTs to HUVECs was assessed by water soluble tetrazolium-8 (WST-8), lactate dehydrogenase (LDH) and neutral red uptake assays. Oxidative stress was indicated by the measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS). ELISA was used to determine the release of inflammatory cytokines. THP-1 monocyte adhesion to HUVECs was also measured. To indicate the activation of endoplasmic reticulum (ER) stress, the expression of ddit3 and xbp-1s was measured by RT-PCR, and BiP protein level was measured by Western blot. Results Transmission electron microscopy observation indicates the internalization of MWCNTs into HUVECs, with a localization in nuclei and mitochondria. The longer MWCNTs induced a higher level of cytotoxicity to HUVECs compared with the shorter ones. Neither of MWCNTs significantly promoted intracellular ROS, but the longer MWCNTs caused a higher depletion of GSH. Exposure to both types of MWCNTs significantly promoted THP-1 adhesion to HUVECs, accompanying with a significant increase of release of interleukin-6 (IL-6) but not tumor necrosis factor α (TNFα), soluble ICAM-1 (sICAM-1) or soluble VCAM-1 (sVCAM-1). Moreover, THP-1 adhesion and release of IL-6 and sVCAM-1 induced by the longer MWCNTs were significantly higher compared with the responses induced by the shorter ones. The biomarker of ER stress, ddit3 expression, but not xbp-1s expression or BiP protein level, was significantly induced by the exposure of longer MWCNTs. Conclusions Combined, these results indicated length dependent toxic effects of MWCNTs to HUVECs in vitro, which might be associated with oxidative stress and activation of ER stress. Electronic supplementary material The online version of this article (10.1186/s12951-017-0318-x) contains supplementary material, which is available to authorized users.

Mesocestoides vogae larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans&mdash, silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)&mdash, on M. vogae larvae at concentrations of 5 and 50 &mu, M under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 &mu, M, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 &mu, M concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism.

Currently, a common therapeutic approach in cancer treatment encompasses a drug combination to attain an overall better efficacy. Unfortunately, it leads to a higher incidence of severe side effects, namely cardiotoxicity. This work aimed to assess the cytotoxicity of doxorubicin (DOX, also known as Adriamycin), 5-fluorouracil (5-FU), cyclophosphamide (CYA), and their combination (5-Fluorouracil + Adriamycin + Cyclophosphamide, FAC) in H9c2 cardiac cells, for a better understanding of the contribution of each drug to FAC-induced cardiotoxicity. Differentiated H9c2 cells were exposed to pharmacological relevant concentrations of DOX (0.13&ndash, 5 &mu, M), 5-FU (0.13&ndash, M), CYA (0.13&ndash, M) for 24 or 48 h. Cells were also exposed to FAC mixtures (0.2, 1 or 5 &mu, M of each drug and 50 &mu, M 5-FU + 1 &mu, M DOX + 50 &mu, M CYA). DOX was the most cytotoxic drug, followed by 5-FU and lastly CYA in both cytotoxicity assays (reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red (NR) uptake). Concerning the equimolar combination with 1 or 5 &mu, M, FAC caused similar cytotoxicity to DOX alone. Even in the presence of higher concentrations of 5-FU and CYA (50 &mu, M CYA), 1 &mu, M DOX was still a determinant for the cardiotoxicity observed in the cytotoxicity assays, phase contrast morphological evaluation, and mitochondrial potential depolarization evaluation. To the best of our knowledge, this was the first in vitro work with this combination regimen, DOX being the most toxic drug and key to the toxicity of FAC.

The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC50 = 0.0037 μM), Nile blue (IC50 = 0.0077 μM) and 1,9-dimethyl methylene blue (IC50 = 0.018 μM) exhibiting higher potency inhibition compared to MB (IC50 = 0.07 μM). Nile blue also represents a potent MAO-B inhibitor with an IC50 value of 0.012 μM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST

Riboflavin (Rf) is an endogenous photosensitizer, which can participate in Type I and Type II processes. We have recently shown that the yield of the triplet excited states of Rf is enhanced in the presence of pectin-coated silver nanoparticles (Pec@AgNP) due to formation of a complex between Rf and Pec@AgNP (Rf-Pec@AgNP). Consequently, under aerobic conditions, the amounts of singlet molecular oxygen and superoxide radical anion generated are also larger in the presence of the nanoparticles. This result made us suspect that the nanoparticles could have a beneficial effect in Rf-based PDT. To prove this hypothesis, we here compared the photodamage in HeLa cells incubated with Rf in the presence and in the absence of Pec@AgNP applying several optical assays. We used fluorescence imaging of irradiated HeLa cells incubated with Annexin V and propidium iodide to evaluate the occurrence of apoptosis/necrosis, the reduction of the tetrazolium dye MTT to formazan and neutral red uptake to prove cell viability, as well as synchrotron infrared microscopy of single cells to evaluate possible structural changes of DNA and nuclear proteins. The enhanced photodamage observed in the presence of Pec@AgNP seems to indicate that Rf enters into the cells complexed with the nanoparticles., Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas, Instituto Multidisciplinario de Biología Celular, Consejo Nacional de Investigaciones Científicas y Técnicas

Natural antioxidants show many pharmacological properties, but poor solubility and inability to cross cell membrane. Liposomes are biocompatible and phospholipid vesicles able to carry hydrophilic, hydrophobic, and amphiphilic molecules. This paper focus on the synthesis of anionic, cationic and zwitterionic liposomes, loaded with quercetin or rutin, and on the evaluation of their cytotoxicity and protective effects against oxidative stress. Chemical characterization was obtained by dynamic light scattering and z-potential experiments. In vitro cell behavior was evaluated by Neutral Red Uptake test. All liposomes, empty and loaded with antioxidants, are stable. The cytotoxicity of both quercetin and rutin encapsulated in zwitterionic and anionic liposomes is higher than that of their solutions. Quercetin and rutin loaded in cationic liposomes are able to inhibit the toxic effect of empty liposomes. The encapsulation of rutin at 5.0×10-5 and 5.0×10-4M, in zwitterionic and anionic liposomes, protects fibroblasts by H2O2 treatment, while the loading with quercetin does not have effect on improving cell viability. All data suggest that the tested liposomes are stable and able to include quercetin and rutin. The liposomes encapsulation of antioxidants makes easier their internalization by cells. Moreover, zwitterionic and anionic liposomes loaded with rutin protect cells by oxidative stress. Liposomes stability together with their good in vitro cytocompatibility, both empty and loaded with antioxidant molecules, makes these systems suitable candidates as drug delivery systems. Moreover, the encapsulation of rutin, is able to protect cells by oxidative stress.

Phenolic compounds play an important role on the growth and reproduction of plants and also exhibit a wide range of pharmacological activities such as anti-allergic, anti-artherogenic, anti-inflammatory and anticancer. But the data about toxicity of many phenolics are lacking and limited. The aim of this study was to evaluate the cytotoxic effects of five different commonly used phenolic compounds (galangin, limonene, naringin, puerarin and ursolic acid) which are widely used because of their health protecting effects by neutral red uptake (NRU) assay in Chinese hamster fibroblast cell line (V79). It is found that all of the studied phenolics decreased the cell viability of V79 cells in a dose dependent manner. The IC50 values of galangin, limonene, naringin, puerarin and ursolic acid were found to be 104.36 pM, 10574 pM, 1976.4 pM, 51.71 pM and 92.94 pM, respectively

This work describes that cytotoxicity of lead chloride and lead acetate to in vitro cultured lymphocytes from human umbilical cord blood, using four monitoring methods namely, trypan blue staining, acridine orange/ethidium bromide staining, 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide (MTT) and neutral red uptake assays; lead genotoxicity to lymphocytes was monitored by comet assay. The MIC value in each method was invariably 300 mg/L for PbCl2. Lethal concentration25 (LC25) values were almost in an agreeable range: 691.83 to 831.76 mg/L; LC50 values in each method were almost in the range: 1174.9 to 1348.9 mg/L; LC100 values were in the range: 3000 to 3300 mg/L, for lead chloride. Similarly, The MIC value in each method were invariably 150 mg/L; LC25 values were almost in the range: 295.12 to 371.53 mg/L; LC50 values were in the range: 501.18 to 588.84 mg/L; LC100 value was 1500 mg/L in all assays, for lead acetate. The comet assay also indicated that the LC100 values were 3300 mg/L lead chloride and 1500 mg/L lead acetate. Thus, both cytotoxicity and genotoxicity were recorded at 3300 mg/L lead chloride and 1500 mg/L lead acetate with lymphocytes.

Made available in DSpace on 2015-10-21T20:58:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-01-01. Added 1 bitstream(s) on 2015-10-22T09:52:42Z : No. of bitstreams: 1 S0102-695X2015000100016.pdf: 1198316 bytes, checksum: 2b987312242bae2e1a5fb1edf85e9f77 (MD5) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) PADC-UNESP A large number of quinones have been associated with antitumor, antibacterial, antimalarial, and antifungal activities. Results of previous studies of 5-methoxy-3,4-dehydroxanthomegnin, a naphthoquinone isolated from Paepalanthus latipes Silveira, Eriocaulaceae, revealed antitumor, antibacterial, immunomodulatory, and antioxidant activities. In this study, we assessed the mutagenicity and metabolism-mediated cytotoxicity of 5-methoxy-3,4-dehydroxanthomegnin by using the Ames test and a microculture neutral red assay incorporating an S9 fraction (hepatic microsomal fraction and cofactors), respectively. We also evaluated the mutagenic activity in Salmonella typhimurium strains TA100, TA98, TA102, and TA97a, as well as the cytotoxic effect on McCoy cells with and without metabolic activation in both tests. Results indicated that naphthoquinone does not cause mutations by substitution or by addition and deletion of bases in the deoxyribonucleic acid sequence with and without metabolic activation. As previously demonstrated, the in vitro cytotoxicity of 5-methoxy-3,4-dehydroxanthomegnin to McCoy cells showed a significant cytotoxic index (CI50) of 11.9 mu g/ml. This index was not altered by addition of the S9 fraction, indicating that the S9 mixture failed to metabolically modify the compound. Our results, allied with more specific biological assays in the future, would contribute to the safe use of 5-methoxy-3,4-dehydroxanthomegnin, compound that has showed in previous studies beneficial properties as a potential anticancer drug. (C) 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. Univ Fed Espirito Santo, Dept Ciencias Farmaceut, BR-29043900 Vitoria, ES, Brazil Univ Estadual Paulista, Inst Quim Araraquara, Araraquara, SP, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut Araraquara, Dept Ciencias Biol, Araraquara, SP, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut Araraquara, Dept Anal Clin, Araraquara, SP, Brazil Univ Estadual Paulista, Inst Quim Araraquara, Araraquara, SP, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut Araraquara, Dept Ciencias Biol, Araraquara, SP, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut Araraquara, Dept Anal Clin, Araraquara, SP, Brazil

International audience; Phycotoxins are monitored in seafood because they can cause food poisonings in humans. Phycotoxins do not only occur singly but also as mixtures in shellfish. The aim of this study was to evaluate the in vitro toxic interactions of binary combinations of three lipophilic phycotoxins commonly found in Europe (okadaic acid (OA), yessotoxin (YTX) and azaspiracid-1 (AZA-1)) using the neutral red uptake assay on two human intestinal cell models, Caco-2 and the human intestinal epithelial crypt-like cells (HIEC). Based on the cytotoxicity of individual toxins, we studied the interactions between toxins in binary mixtures using the combination index-isobologram equation, a method widely used in pharmacology to study drug interactions. This method quantitatively classifies interactions between toxins in mixtures as synergistic, additive or antagonistic. AZA-1/OA, and YTX/OA mixtures showed increasing antagonism with increasing toxin concentrations. In contrast, the AZA-1/YTX mixture showed increasing synergism with increasing concentrations, especially for mixtures with high YTX concentrations. These results highlight the hazard potency of AZA-1/YTX mixtures with regard to seafood intoxication.

Four previously undescribed diterpenoids including two crotofolanes, crotodichogamoin A and B, and two halimanes, crothalimene A and B, a new sesquiterpenoid, and fifteen previously reported compounds, including the crotofolane, crotohaumanoxide, the casbane, depressin, a further seven furanohalimane diterpenoids, three patchoulane and two further cadinane sesquiterpenoids and aleuritolic acid were isolated from the root of Croton dichogamus. Crotodichogamoin B is an important biosynthetic intermediate of the crotofolane class and this is the first report of patchoulene sesquiterpenoids from the genus. Compounds were tested at one concentration, 1x10-5 M, in the NCI59 cell one-dose screen but did not show significant activity snd were also evaluated for their cytotoxicity against Caco-2 cell lines using the neutral red assay. 10-epi-Maninsigin D reduced Caco-2 cell viability at 10, 30 and 100 µM, with values of decreased viability of 28%, 48% and 43% respectively. None of the other tested compounds showed significant activity.

For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS) and its bioactive constituent protocatechuic acid (PCA), have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS)-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC50 values of 0.92 and 1.43 µg∙mL−1, respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC50 value of 82.4 µg∙mL−1. This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.

New derivatives of N-[(phenoxy)alkyl]- and N-[(phenoxy)ethoxyethyl]aminoalkanols have been synthesized and evaluated for their anticonvulsant activity in maximal electroshock (MES), maximal electroshock seizure threshold (MEST), and pentylenetetrazol (PTZ) tests. Their neurotoxicity was evaluated via rotarod and chimney tests. The compounds exhibiting the most beneficial activity and protection indices were evaluated for analgesic activity using the formalin test for neurogenic pain. They were also evaluated for their influence on cytotoxic activity using in vitro cellular models (HepG2 and CRL-2534 cell lines). Experiments performed using MTT and neutral red cytotoxicity assays showed that all evaluated compounds were safe for normal, glial cells (astrocytes) and did not induce hepatotoxic effects. Based on the results from the in vitro studies, the safety of the evaluated compounds was inferred. The most promising compound in this research was 1-{2-[2-(2,3-dimethylphenoxy)ethoxy]ethyl}piperidin-3-ol hydrochloride. Additionally, in silico metabolism prediction for the compound has been performed.

WOS: 000405251800006, Malignancies constitute a global health concern and chemotherapy remains the main mode of treatment. The present study was designed to evaluate the cytotoxicity of 8 compounds from Desbordesia glaucescens namely lanosta-7,24-dien-3-one (1), friedelanone (2), friedelanol (3), 3,3'-di-O-methylellagic acid (4), 3,3',4'-tri-0-methylellagic acid (5), ellagic acid (6), 3',4'-di-0-methylellagic acid 4-0-beta-o-glucopyranoside (7) and 3,3'-di-0-methylellagic acid 4'-0-beta-c-xylopyranoside (8) against 4 human carcinoma cell lines and normal CRL2120 fibroblasts. The neutral red uptake (NRU) assay was used for cytotoxicity testing. Caspase-Glo assay, cell cycle analysis, measurements of mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were used to evaluate apoptosis induction. Compounds 4 and 6 as well as doxorubicin had IC50 values below 45 pM in the four tested cancer cell lines meanwhile other compounds displayed selective activity. The IC50 values ranged from 11.23 mu M (towards breast adenocarcinoma MCF-7 cells) to 44.65 mu M (colon carcinoma Caco-2 cells) for 4, from 14.07 mu M (towards MCF-7 cells) to 77.73 mu M (Caco-2 cells) for 6 and from 0.07 mu M (towards SPC212 cells) to 1.01 mu M (A549 cells) for doxorubicin. Compound 4 induced apoptosis in MCF-7 cells mediated by MMP loss. The constituents of Desbordesia glaucescens and especially ellagic acid (6) and its derivative 4 are potential cytotoxic compounds that deserve more investigations towards developing novel antiproliferative drugs against human carcinoma., Scientific and Technological Research Counsel of Turkey (TUBITAK); Scientific Research Projects Commission of Anadolu University, Eskisehir, Turkey [1507F563, 1306F110]; International Science Programme, Uppsala University, Sweden (ISP) [KEN-02], V.K. and H.S. are thankful to Scientific and Technological Research Counsel of Turkey (TUBITAK) for 6 months travel grant (to V.K.) and to Scientific Research Projects Commission of Anadolu University, Eskisehir, Turkey for the funding grant 1507F563 (to V.K. and H.S.). A grant for part of this work was also provided by International Science Programme, Uppsala University, Sweden (ISP)-KEN-02 project. IC would like to thank the Scientific Research Projects Commission of Anadolu University, Eskisehir, Turkey for the funding grant (1306F110). Authors are also thankful to Sennur Gorgulti for FACS measurements.

In this study, four polysaccharide fractions designated as RGP1, RGP2, RGP3, and RGP4 were isolated from red ginseng by DEAE-52 cellulose chromatography, and their macrophage immunomodulatory activities were investigated. The results revealed that the proliferation, NO production, and neutral red phagocytosis of RAW 264.7 macrophage cells in groups treated with RGP1 and RGP2 in vitro were increased significantly compared to RGP3 and RGP4. In addition, the level of TNF-α in RAW 264.7 cells was significantly increased in RGP1 and RGP2 groups. All the results consistently indicated that polysaccharide fractions RGP1 and RGP2 had strong macrophage immunomodulatory activities. Furthermore, RGP1 and RGP2 were purified by Sephadex G-100 column and RGP2 was further fractionated into a homogeneous fraction RGP2-1, with the molecular weight of 2.16 × 104 Da. The analysis of monosaccharide composition revealed that RGP1 was composed of arabinose, glucose, and galactose with a relative molecular ratio of 0.02 : 0.88 : 0.10. RGP2-1 was composed of rhamnose, arabinose, glucose, and galactose with a relative molecular ratio of 0.02 : 0.10 : 0.77 : 0.11. These results provided evidences that the neutral polysaccharide fractions RGP1 and RGP2 possessed significant immunomodulatory activity and could be explored as a promising natural immunomodulating agent applied in functional foods or medicines.

Coleostephus myconis (L.) Rchb.f. (Asteraceae) is a highly disseminated plant species with ruderal and persistent growth. Owing to its advantageous agronomic properties, C. myconis might have industrial applications. However, this species needs to be comprehensively characterized before any potential use. In a previous study, the phenolic composition and antioxidant activity of different C. myconis tissues were characterized. This investigation was extended to examine the cytotoxic potential of selected plant tissues (flowers and green parts) using a HepG2 cell line by utilizing the lysosomal neutral red uptake assay or mitochondrial (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. In addition, the macronutrients content, lipophilic compounds (fatty acids, tocopherols), and amino acids were also determined. C. myconis flowers were used in the senescence stage, which was previously identified as the stage that presented maximal phenolic content and highest antioxidant activity. In contrast, stems and leaves were employed due to their high biomass proportion. Regarding cytotoxicity, mitochondrial and lysosomal damage was only significant when HepG2 cells were exposed to the highest extract concentrations (stems and leaves, 0.9 mg/ml; senescent flowers, 0.3 mg/ml). Chemically, the senescent flowers were mostly characterized by their high levels of fat, amino acids (especially threonine), oleic acid, ß-, and ?-tocopherol, while stems and leaves contained high concentrations of carbohydrates, linolenic acid, and a-tocopherol. In general, these results provide information regarding the threshold concentrations of C. myconis extracts that might be used in different applications without toxicity hazards. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support to REQUIMTE (PEst-C/EQB/LA0006/2014) and to CIMO (UID/AGR/00690/2013). J.C.M. Barreira, Carla Costa, and Filipa B. Pimentel thank FCT, POPH-QREN, and FSE for their grants (SFRH/BPD/72802/2010, SFRH/BPD/96196/2013 and SFRH/BD/109042/2015, respectively). info:eu-repo/semantics/publishedVersion

International audience; Poly(HexaMethylene Biguanide) hydrochloride (PHMB) CAS No. [32289-58-0] is a particularly effective member of the biguanides antiseptic chemical group, and has been in use since the early fifties in numerous applications. It has been proposed that PHMB be classified as a category 3 carcinogen although PHMB is not genotoxic. It has been hypothesized that PHMB may have epigenetic properties effects, including non-genotoxic modifications of DNA bases, DNA methylation and mitogenic cytokine production. These properties have been assessed in vitro using 3 cell types: Caco-2 cells (from a human colon adenocarcinoma) with a non-functional p53 gene. (∆p53: mut p53), N2-A (Neuro-2A cells, mouse neural cells), the brain being a possible target organ in rodents and HepG2 cells (human hepatocellular carcinoma) with functional p53 gene. From the concentration 1 µg/mL up to 20 µg/mL of PHMB, no effect was observed, either growth stimulation or inhibition. Viability testing using neutral red led to an IC 50 of 20-25 µg/mL after treatment with PHMB for 3 h, whereas the MTT test led to IC50 values of 80 µg/mL, 160 µg/mL and 160 µg/mL respectively for HepG2 cells, Neuro-2A cells and Caco-2 cells. PHMB does not induce significant oxidative stress (production of MDA or lipoperoxidation, nor does it induce hydroxylation of DNA (8-OH-dG) and/or its hypermethylation (m5dC), the latter being strongly implicated in DNA replication and regulation and cell division. PHMB does not induce significant production of mitogenic cytokines such as TNF-α (tumor necrosis factor), interleukins (IL-1 alpha), and the transcription factor nuclear factor kappa B (NF-κB) which can cause either apoptosis or stimulate the growth of transformed cells or tumors. Instead, from concentrations of 20 to 100 µg/mL, PHMB kills cells of all types in less than 3 h. The expression of genes involved in the mechanisms of cell death induced by PHMB, including p53, the pro apoptotic gene bax and others, the anti-apoptotic bcl-2 and caspase-3 has been evaluated by RT-PCR. Finally, the status of GAP-junctions (GJIC) in the presence of PHMB has been determined and appeared to not be significantly affected. Taken together the data show that in vitro PHMB does not exhibit clear and remarkable epigenetic properties except a slight increase of some cytokines and transcription factor at higher concentrations at which cell lysis occurs rapidly.

Purpose: To investigate the protective effects of Portulaca oleracea seed extract (POA) against cytotoxicity, oxidative stress and reactive oxygen species (ROS) generation induced by hydrogen peroxide (H 2 O 2 ) in human liver cells (HepG2). Methods: The extract (POA) was obtained by ethanol extraction of P. oleracea seeds. Cytotoxicity in HepG2 cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, neutral red uptake (NRU) assay and morphological changes. The cells were pre-exposed to noncytotoxic concentrations (5 - 25 μg/mL) of POA for 24 h, and then cytotoxic (0.25 mM) concentration of H 2 O 2 . After 24 h of exposure, MTT and NRU assays were used to evaluate cell viability, while morphological changes were assessed using phase contrast inverted microscopy. The effect of POA on reduced glutathione (GSH) level, lipid peroxidation (LPO), and ROS generation induced by H 2 O 2 was also studied. Results: The results showed that pre-exposure to POA (25 μg/mL) significantly (p

Nickel nanomaterials are promising in the biomedical field, especially in cancer diagnostics and targeted therapy, due to their distinctive chemical and physical properties. In this experiment, the toxicity of nickel nanotubes (Ni NTs) were tested in an in vitro cervical cancer model (HeLa cell line) to optimize the parameters of photodynamic therapy (PDT) for their greatest effectiveness. Ni NTs were synthesized by electrodeposition. Morphological analysis and magnetic behavior were examined using a Scanning electron microscope (SEM), an energy dispersive X-ray analysis (EDAX) and a vibrating sample magnetometer (VSM) analysis. Phototoxic and cytotoxic effects of nanomaterials were studied using the Ni NTs alone as well as in conjugation with aminolevulinic acid (5-ALA); this was performed both in the dark and under laser exposure. Toxic effects on the HeLa cell model were evaluated by a neutral red assay (NRA) and by detection of intracellular reactive oxygen species (ROS) production. Furthermore, 10–200 nM of Ni NTs was prepared in solution form and applied to HeLa cells in 96-well plates. Maximum toxicity of Ni NTs complexed with 5-ALA was observed at 100 J/cm2 and 200 nM. Up to 65–68% loss in cell viability was observed. Statistical analysis was performed on the experimental results to confirm the worth and clarity of results, with p-values = 0.003 and 0.000, respectively. Current results pave the way for a more rational strategy to overcome the problem of drug bioavailability in nanoparticulate targeted cancer therapy, which plays a dynamic role in clinical practice.

Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10, in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013-2014 PM10 was sampled (24 h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The reduction in cell viability was significantly correlated with BC, Cd and Pb. The induction of IL-8 in Beas-2B cells was significantly associated with Cu, Ni and Zn and endotoxin. Endotoxin levels explained 33% of the variance in IL-8 induction. A significant interaction between ambient temperature and endotoxin on the pro-inflammatory activity was seen. No association was found between OP and the cellular responses. This study supports the hypothesis that, on an equal mass basis, PM10 induced biological effects differ due to differences in PM10 characteristics. Metals (Cd, Cu, Ni and Zn), BC, and endotoxin were among the main determinants for the observed biological responses. (c) 2016 Elsevier Inc. All rights reserved.

Oxidative stress in the small intestinal epithelium can lead to barrier malfunction. In this study, the effect of rosmarinic acid (RA), quercetin (Que), gallic acid (GA), lipoic acid (LA), ethoxyquin (ETQ) and Se-methionine (SeMet) pre-treatments using 2 mM Trolox as a control on the viability and the generation of intracellular reactive oxygen species (iROS) of oxidatively (H2O2) stressed intestinal porcine epithelial cells (IPEC-J2) was investigated. A neutral red assay showed that RA (50-400 mu M), Que (12.5-200 mu M), GA (50-400 mu M), ETQ (6.25-100 mu M), and SeMet (125-1000 mu M) pre-treatments but not LA significantly increased the viability of H2O2-stressed IPEC-J2 cells (p < 0.05). A 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA) fluorescent probe showed that RA (100-600 mu M), Que (25-800 mu M), ETQ (3.125-100 mu M) and SeMet (500-2000 mu M) pre-treatments significantly reduced iROS in IPEC-J2 monolayers (p < 0.05). Moreover, RA and Que were most effective in reducing iROS. Therefore, the effects of RA and Que on barrier functioning in vitro were examined. RA and Que pre-treatments significantly decreased fluorescein isothiocyanate (FITC)-conjugated dextran-4 (4 kDa) permeability and transepithelial electrical resistance (TEER) of an IPEC-J2 cell monolayer (p < 0.05). These in vitro results of RA and Que hold promise for their use as antioxidants in pig feed.

The ethanol extract of the vegetal species Pentaclethra macroloba (Willd.) Kuntze, Fabaceae, was fractioned and the antibacterial activity was determined. The active ethyl acetate (ea) fraction showed activity against Gram-positive (Staphylococcus spp. and Enterococcus spp.) and Gram-negative (Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella pneumoniae) multiresistant bacteria. Gallic acid derivatives were identified as the main compounds in inactive subfractions from the ea fraction, while the active one afforded ellagic acid as the major constituent when submitted to acid hydrolysis reaction, which suggests the presence of hydrolysable tannins. The minimum bactericidal concentration analysis showed a bactericide mechanism of action for the tannin subfraction found. The antibacterial mechanism of action of the active tannin subfraction against S. aureus reference strains (ATCC 29213 e 33591) was proposed adopting an in vitro assay of protein synthesis inhibition. For this, bacterial cells were labeled with [35S] methionine in the presence of the subfraction. The protein synthesis inhibition was observed at 256 µg/mL of this subfraction. At this concentration it did not present cytotoxicity in eukaryotic cells by the neutral red technique, suggesting selective toxicity. The present study is the first in vitro investigation of the antibacterial properties of tannin fractions obtained from a polar extract of P. macroloba.

Human 15-lipoxygenase-1 (h-15-LOX-1) is a promising drug target in inflammation and cancer. In this study substitution-oriented screening (SOS) has been used to identify compounds with a 2-aminopyrrole scaffold as inhibitors for h-15-LOX-1. The observed structure activity relationships (SAR) proved to be relatively flat. IC50's for the most potent inhibitor of the series did not surpass 6.3 μM and the enzyme kinetics demonstrated uncompetitive inhibition. Based on this, we hypothesized that the investigated 2-aminopyrroles are pan assay interference compounds (PAINS) with photoactivation via a radical mechanism. Our results demonstrated clear photoactivation of h-15-LOX-1 inhibition under UV and visible light. In addition, the investigated 2-aminopyrroles decreased viability of cultured human hepatocarcinoma cells HCC-1.2 in a dose-dependent manner with LD50 ranging from 0.55 ± 0.15 μM (21B10) to 2.75 ± 0.91 μM (22). Taken together, this indicates that photoactivation can play an important role in the biological activity of compounds with a 2-amino-pyrrole scaffold as investigated here.

Texto completo: acesso restrito. p.387–396 Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2015-03-03T13:04:07Z No. of bitstreams: 1 22- Environ. Res. 99 p. 387–396, 2005.pdf: 322523 bytes, checksum: 483b05147568729ec2094af5573e9901 (MD5) Approved for entry into archive by Alda Lima da Silva (sivalda@ufba.br) on 2015-03-03T14:17:50Z (GMT) No. of bitstreams: 1 22- Environ. Res. 99 p. 387–396, 2005.pdf: 322523 bytes, checksum: 483b05147568729ec2094af5573e9901 (MD5) Made available in DSpace on 2015-03-03T14:17:50Z (GMT). No. of bitstreams: 1 22- Environ. Res. 99 p. 387–396, 2005.pdf: 322523 bytes, checksum: 483b05147568729ec2094af5573e9901 (MD5) Previous issue date: 2005 Coastal marine environments, especially semienclosed systems such as bays, are under unrelenting stress caused by urban and industrial development. Biomonitoring plays a vital role in strategies to identify, assess, and control stressors. However, due to the magnitude of the challenge there is a demand for new and innovative approaches to provide timely and accessible information to environmental managers and policy makers. The present work aimed to assess hydrocarbon levels in sediments from petroleum-related industrial areas at Todos os Santos Bay (Brazil) and associate them to toxicity-induced responses (neutral red retention (NRR) assay) by the burrowing clam Anomalocardia brasiliana. Surface sediments collected during the dry and rainy seasons were analyzed for aliphatic and aromatic hydrocarbons. At the control site, hydrocarbon levels were low and mainly biogenic. The aliphatic hydrocarbon (“total unresolved complex mixture,” alkanes, and isoprenoids) concentrations indicated a chronic situation with very little “fresh” oil contamination at the oil-related sites. The polycyclic aromatic hydrocarbons indicated sites moderately contaminated by chronic oil and some pyrolytic input. The effects of those contaminants were assessed by the lysosomal NRR assay applied to A. brasiliana hemocytes. Sediment toxicity at the oil-related sites was evidenced by the lowered capacity of the lysosomes to retain the neutral red dye compared to results from the control site. This research indicates that the NRR assay is a useful and efficient screening technique able to discriminate polluted from clean sites.

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.

Exopolyphosphatase activity and vacuolar acidity were assessed in order to have an overview of regulation of polyphosphate metabolism in arbuscular mycorrhizal symbiosis. Marigold (Tagetes patula) was inoculated with Glomus coronatum or G. etunicatum and grown in a mesh bag system. The extraradical hyphae were harvested from outside the mesh bag and the intraradical hyphae were collected from the mycorrhizal roots after enzymic digestion. Exopolyphosphatase activity was measured at pH 7.5 and 5.0. The extra- and intraradical hyphae of G. coronatum showed activity at pH 7.5 and 5.0, respectively. The extraradical hyphae of G. etunicatum showed activity both at pH 7.5 and 5.0, whereas intraradical hyphae of this fungus showed activity only at pH 5.0. The neutral (pH 7.5) activity showed lower Km with long-chain polyphosphate, whereas the acidic (pH 5.0) activity showed lower Km with short-chain polyphosphate. The hyphae were stained with Neutral red, an acidic compartment probe, because a pH gradient across the tonoplast has been thought to be essential for polyphosphate accumulation in vacuoles. Both extra- and intraradical hyphae had acidic compartments which were identified as vacuoles. The inoculated plants were grown for 5 weeks with P-fertilizer (50 μM KH2PO4). P was then withheld from half of the plants, to provide low P and +P treatments. After 1 week, polyphosphate in the hyphae was stained by Toluidine blue. Polyphosphate was found in the hyphae from the +P treatment but not in the hyphae from the low P treatment. In contrast, exopolyphosphatase activity and acidity of vacuoles were relatively constant irrespective of polyphosphate status. Based upon these observations it is concluded that the fungi have at least two different exopolyphosphatase-type enzymes which are differently expressed between extra- and intraradical hyphae and that polyphosphate accumulation may be dynamically regulated by the balance between synthesis and hydrolysis.

9 pages, 2 figures, 2 tables.-- PMID: 15033545 [PubMed].-- Printed version published May 3, 2004., Extensive efforts have been made, recently, to find surfactants with lower irritation potential than those presently commercially available, for use in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of a novel family of dicationic arginine-diglyceride surfactant compounds, 1,2-diacyl,3-O-(Image -arginyl)-rac-glycerol with alkyl chain lengths in the range from 8 to 14 carbon atoms, were compared to three commercial surfactants. The end-points used to assess toxicity were the red blood cell lysis assay and uptake of the vital dye neutral red 24 h after dosing (NRU), respectively. Two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, were used as in vitro models to predict the potential phototoxicity which could result in irritation, determined by resazurin reduction to resorufin and neutral red uptake (NRU). All tested surfactants had cytotoxicity effects as demonstrated by and decrease of NR uptake, which showed a clear concentration-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC50) range from 1 μmol l−1 (hexadecyl trimethyl ammonium bromide) to 565 μmol l−1 (12,12-Image -arginine). Erythrocyte haemolysis also showed a clear concentration–response relationship, the 50% of haemolysis ranged from 37 μmol l−1 (10,10-Image -arginine) to 151 μmol l−1 (sodium lauryl sulphate). Phototoxicity was performed with 12,12-Image -acetyl-arginine, the most stable chemical structure. The validated 3T3 NRU photoxicity assay was used and revealed a phototoxic potential., This research was supported by the project PPQ2000-1687-C02-01 from CICYT (Spain).