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The neutral red uptake (NRU) assay is a cell viability assay that can be used for the assessment of compound-induced cytotoxicity. It is based on the ability of living cells to incorporate neutral red, a weak cationic dye, in lysosomes. The quantification of xenobiotic-induced cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red when compared to cells exposed to corresponding vehicle controls. The NRU assay is mainly used for hazard assessment in in vitro toxicology applications. Hence, this method has been incorporated in regulatory recommendations such as the OECD test guideline TG 432, in which an in vitro 3T3-NRU-phototoxicityassay is described to assess the cytotoxicity of compounds in the presence or absence of UV light.This book chapter describes a detailed protocol to carry out the NRU assay using the human hepatoma cell line HepG2, which is frequently employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetylsalicylic acid is assessed., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy., (© 2022 Wiley Periodicals LLC.)

Numerous studies have reported on the positive effects of silicon (Si) on bone metabolism, particularly on the stimulatory effects of Si on osteoblast cells and on bone formation. Inhibitory effects of Si on osteoclast formation and bone resorption have also been demonstrated in vitro and are suggested to be mediated indirectly via stromal and osteoblast cells. Direct effects of Si on osteoclasts have been less studied and mostly using soluble Si, but no characterisation of the Si treatment solutions are provided. The aims of the present study were to (a) further investigate the direct inhibitory effects of Si on osteoclastogenesis in RANKL-stimulated RAW264.7 cells, (b) determine at what stage during osteoclastogenesis Si acts upon, and (c) determine if these effects can be attributed to the biologically relevant soluble orthosilicic acid specie. Our results demonstrate that silicon, at 50 μg/ml (or 1.8 mM), does not affect cell viability but directly inhibits the formation of TRAP+ multinucleated cells and the expression of osteoclast phenotypic genes in RAW264.7 cells. The inhibitory effect of Si was clearly associated with the early stages (first 24 hr) of osteoclastogenesis. Moreover, these effects can be attributed to the soluble orthosilicic acid specie., (© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Marine-derived fungi proved to be a rich source of biologically active compounds. The genus Penicillium has been extensively studied regarding their secondary metabolites and biological applications. However, the photoprotective effects of these metabolites remain underexplored. Herein, the photoprotective potential of Penicillium echinulatum, an Antarctic alga-associated fungus, was assessed by UV absorption, photostability study, and protection from UVA-induced ROS generation assay on human immortalized keratinocytes (HaCaT) and reconstructed human skin (RHS). The photosafety was evaluated by the photoreactivity (OECD TG 495) and phototoxicity assays, performed by 3T3 neutral red uptake (3T3 NRU PT, OECD TG 432) and by the RHS model. Through a bio-guided purification approach, four known alkaloids, (-)-cyclopenin (1), dehydrocyclopeptine (2), viridicatin (3), and viridicatol (4), were isolated. Compounds 3 and 4 presented absorption in UVB and UVA-II regions and were considered photostable after UVA irradiation. Despite compounds 3 and 4 showed phototoxic potential in 3T3 NRU PT, no phototoxicity was observed in the RHS model (reduction of cell viability < 30%), which indicates their very low acute photoirritation and high photosafety potential in humans. Viridicatin was considered weakly photoreactive, while viridicatol showed no photoreactivity; both compounds inhibited UVA-induced ROS generation in HaCaT cells, although viridicatol was not able to protect the RHS model against UVA-induced ROS production. Thus, the results highlighted the photoprotective and antioxidant potential of metabolites produced by P. echinulatum which can be considered a new class of molecules for photoprotection, since their photosafety and non-cytotoxicity were predicted using recommended in vitro methods for topical use., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 CC 50 values for both cell lines were comparable with analogous IC 50 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.

Phototoxicity due to dermally impregnated compounds exposed to ultraviolet radiation is associated with skin inflammation. The phototoxicity potential of active substances applied to the skin should be evaluated. Pigments are widely used for tattoos, and hypersensitivity reactions, such as photoallergic dermatitis, are possible tattoo-related complications. However, the phototoxicity of these chemicals is not well known. In this study, we evaluated the phototoxicity potential of six tattoo pigments, cadmium sulfide, carbazole, cadmium selenide, mercury (II) sulfide, chromium oxide, and cobalt aluminate, using in vitro methods-3 T3 neutral red uptake (NRU) phototoxicity test (PT) and a 3D human reconstructed skin model (EpiDerm). The validated 3 T3 NRU PT indicated the phototoxicity potential of carbazole and cadmium sulfide. The 3D human skin model confirmed that only carbazole was phototoxic. The 3 T3 NRU PT data corresponded well with those from the 3D skin model and suggested the need to employ several test systems for final phototoxicity assessment. In addition to the results obtained using 3 T3 NRU PT, further testing on 3D skin models may better reflect the bioavailability of a given chemical in the skin., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Lycopene is one of the most potent antioxidants among carotenoids due to its ability to quench singlet oxygen and react with free radicals to reduce DNA damage. Methotrexate is widely used in the treatment of several types of cancers and autoimmune diseases. One of the most common side effects of a high-dose of methotrexate is kidney injury. In this study, we evaluated effects of lycopene on the Madin-Darby canine kidney cells (MDCK) treated with methotrexate through the estimation of their mitochondrial and lysosomal functions ((4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay and neutral red uptake assay) and changes in cell oxidative status (determination of advanced oxidized proteins concentrations and reduced glutathione levels) and lysosomal enzymes activity (β- N -acetyl glucosaminidase activity). Results of our study showed that lycopene applied in high concentration caused significant impairment of the MDCK function leading to cell death. Contrarily, in relatively low concentrations lycopene moderately ameliorated methotrexate-induced MDCK cell death estimated by both biochemical and microscopic analyses. It also prevented a significant decline in the MDCK cell lysosomal function estimated by neutral red accumulation ability and activity of the lysosomal enzyme β- N -acetyl glucosaminidase.

The lack of predictivity of animal's models has increased the failure rate of drug candidates. Thus, the reversion of this scenario using preliminary in vitro assays and metabolism prediction can reduce the unnecessary use of animals, as well as predict toxic effects at preclinical and clinical stages. The present study aimed to evaluate safety of four biologically active molecules (RN104, RI78, ICH, PCH) with potential therapeutic applications synthesized in our laboratory. Initially, we used MTT cytotoxicity against A549, H9C2, HepG2, LLC-PK1 and NEURO-2 cell lines. RN104 showed the lowest cytotoxicity and further studies were conducted with it. The neutral red (NR) test was performed according to OECD-129 and then acute toxicity test (OECD-423). According to NR results we administered at 300 mg/kg on animals; however, no toxic effect was observed, while 2,000 mg/kg resulted in the death of one animal per group. After, metabolism prediction studies, performed using both ligand-based and structure-based, suggests three potential metabolites. In silico results suggested that potential metabolites could be fast eliminated and, then, this could be an explanation for lower observed toxicity in in vivo experiments. The results showed limitations of the NR as a predictor of the initial dose for the acute toxicity study, which may be related to metabolism. Therefore, the combination of theoretical and experimental studies is relevant to a general understanding of new molecule's toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

Cytotoxicity assays are used to quantify the cytotoxic potential of chemicals. The neutral red uptake (NRU) assay is one of these assays and is routinely used in the pharmaceutical, cosmetic, and tobacco industries. In the context of e-cigarette development, an NRU assay-based screen was implemented to evaluate the cytotoxic potential of e-liquids. E-liquids induced a biphasic response in the BALB/c 3T3-based assay. The NRU initially increased in a concentration-dependent manner before decreasing following treatment with higher concentrations until NRU was abolished. Experiments were performed to characterize the mechanism underlying this biphasic signal. Nicotine alone was found to induce the same biphasic effects, while inducing concentration-dependent decreases in relative cell counts (RCC). Imaging and flow cytometry data revealed that the increases in NRU likely resulted from nicotine-induced vacuolization via a lysosomotropic mechanism. In support of this, two lysosomotropic agents, chloroquine and lapatinib, induced similar profiles. Nicotine's effects were also translatable, as brain-, lung-, bone marrow-, and smooth muscle-derived mammalian cells responded with the biphasic NRU signal. However, like RCC, three other cytotoxicity endpoints, resazurin, adenosine triphosphate, and water soluble tetrazolium salt (WST)-8, were not subject to these effects. The WST-8 assay is proposed as an alternative to screen the cytotoxic potential of e-liquids., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To assess photoxicity, several in vitro methods using different cellular models have been developed for preclinical testing. Over prediction of the in vivo photosafety hazard has been however appointed. Herein, we describe the implementation and validation of an in vitro methodology for phototoxicity evaluation based on the 3T3 neutral red uptake phototoxicity test using the HaCaT human keratinocyte cell line, and UVA/UVB radiation. Known positive (5-methoxypsoralen, chlorpromazine, and quinine) and negative (acetyl salicylic acid, hexachlorophene, and sodium lauryl sulphate) controls were tested together with a set of chemical currently used in cosmetic/pharmaceutical formulations. Apart from the advantage of using a cell line of human origin, these cells were generally more resistant to the cytotoxic effects of the test substances relative to the 3T3 mouse fibroblasts when exposed to an UVA irradiation dose of 1.7 mW/cm 2 . Therefore, this HaCaT NRU assay provides a more realistic experimental model that overcomes the over/high sensitivity frequently noted with the 3T3 NRU assay and that is more consistent with the human in vivo situation. Using a more representative method can prevent time-consuming and expensive in vivo testing in both animal models and humans that can significantly delay the clinical development of new chemicals., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

One of the most appreciated capabilities of computational toxicology is to support the design of pharmaceuticals with reduced toxicological hazard. To this end, we have strengthened our drug photosafety assessments by applying novel computer models for the anticipation of in vitro phototoxicity and human photosensitization. These models are typically used in pharmaceutical discovery projects as part of the compound toxicity assessments and compound optimization methods. To ensure good data quality and aiming at models with global applicability we separately compiled and curated highly chemically diverse data sets from 3T3 NRU phototoxicity reports (450 compounds) and clinical photosensitization alerts (1419 compounds) which are provided as supplements. The latter data gives rise to a comprehensive list of explanatory fragments for visual guidance, termed phototoxophores, by application of a Bayesian statistics approach. To extend beyond the domain of well sampled fragments we applied machine learning techniques based on explanatory descriptors such as pharmacophoric fingerprints or, more important, accurate electronic energy descriptors. Electronic descriptors were extracted from quantum chemical computations at the density functional theory (DFT) level. Accurate UV/vis spectral absorption descriptors and pharmacophoric fingerprints turned out to be necessary for predictive computer models, which were both derived from Deep Neural Networks but also the simpler Random Decision Forests approach. Model accuracies of 83-85% could typically be reached for diverse test data sets and other company in-house data, while model sensitivity (the capability of correctly detecting toxicants) was even better, reaching 86%-90%. Importantly, a computer model-triggered response-map allowed for graphical/chemical interpretability also in the case of previously unknown phototoxophores. The photosafety models described here are currently applied in a prospective manner for the hazard identification, prioritization, and optimization of newly designed molecules.

The pollution by industrial and municipal effluents are major sources of concerns. Fish cell cultures were applied in different strategies of the evaluation of effluents, particularly whole toxicity, toxicity identification evaluation and mode of action studies based in adverse outcome pathways. Whole effluent toxicity was evaluated using a battery of five model systems from four trophic levels: Daphnia magna was the most sensitive system, followed by the hepatoma fish cell line PLHC-1, the bacterium Allivibrio fischeri, the fibroblastic fish cell line RTG-2 and the algae Chlorella vulgaris, detecting a risk of eutrofization. The uptake of neutral red was more sensitive than the content of protein assay. The main morphological alterations observed were cell loss, hydropic degeneration, and a general loss of lysosomes and of their perinuclear distribution. The toxicity was characterized in PLHC-1 cells through toxicity identification evaluation, in which a partial reduction with graduation at pH 11, filtration, aeration and addition of thiosulfate or EDTA was shown; on the other hand, a low sorption in solid phase extraction suggested that the main responsible were not organic compounds. Consequently, it was not necessary to apply an effect directed analysis HPLC fractionation. In the chemical identification phase, Zn, Cd, As, Cu and Pb were quantified in decreasing concentrations. In the toxicity confirmation phase, a reconstituted sample and individual solutions, presented decreasing toxicity: Zn > Pb > As +5  > Cd > Cu > As +3 , the global toxicity being explained by response addition. In the last step, the mode of action was investigated using five specific biomarkers. While metallothionein and succinate dehydrogenase activity were increased, no changes occurred for lysosomal function, acetylcholinesterase and EROD activities, the responsibility of the toxicity for the elements found being confirmed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Microbial electrosynthetic cells containing Methylobacterium extorquens were studied for the reduction of CO 2 to formate by direct electron injection and redox mediator-assisted approaches, with CO 2 as the sole carbon source. The formation of a biofilm on a carbon felt (CF) electrode was achieved while applying a constant potential of -0.75 V versus Ag/AgCl under CO 2 -saturated conditions. During the biofilm growth period, continuous H 2 evolution was observed. The long-term performance for CO 2 reduction of the biofilm with and without neutral red as a redox mediator was studied by an applied potential of -0.75 V versus Ag/AgCl. The neutral red was introduced into the systems in two different ways: homogeneous (dissolved in solution) and heterogeneous (electropolymerized onto the working electrode). The heterogeneous approach was investigated in the microbial system, for the first time, where the CF working electrode was coated with poly(neutral red) by the oxidative electropolymerization thereof. The formation of poly(neutral red) was characterized by spectroscopic techniques. During long-term electrolysis up to 17 weeks, the formation of formate was observed continuously with an average Faradaic efficiency of 4 %. With the contribution of neutral red, higher formate accumulation was observed. Moreover, the microbial electrosynthetic cell was characterized by means of electrochemical impedance spectroscopy to obtain more information on the CO 2 reduction mechanism., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The current study is based on the increasing demand for the assessment of ionic liquid (IL)-mediated aquatic toxicity. Specifically, although a lot of studies have been performed so far, investigating IL-mediated adverse effects on numerous aquatic organisms, little is known about their mode of action. Given that the use of in vitro models is considered as a reliable tool for determining the mediated biological effects, the modulation of specific biochemical pathways and the onset of various forms of damage with great precision and reproducibility, mixed primary cultures of mussel Mytilus galloprovincialis hemocytes were used for investigating whether 1-octyl-3-methylimidazolium tetrafluoroborate ([omim][BF 4 ]) mediated toxicity is related to its interaction with cellular membrane proteins. Specifically, [omim][BF 4 ]-mediated cytotoxic, oxidative and genotoxic effects were investigated in mussel hemocytes before and after pre-treatment of cells with non-toxic concentration of guanidine hydrochloride (1 mM GndHCl). The results showed that [omim][BF 4 ] at concentrations ranging from 0.7 to 1.75 μM can induce cytotoxic (almost <50% reduction of cell viability), oxidative (increased levels of O 2 •- production and lipid peroxidation by-products) and genotoxic (increased levels of DNA damage) effects, while cells pre-treated with 1 mM GndHCl showed a significant attenuation of IL's toxic potency in all cases. According to the latter, the current study showed that [omim][BF 4 ]-mediated toxicity could be related not only to its well-known interaction with membrane lipid bilayers, but also to its interference with membrane proteins. Using GndHCl, a chaotropic agent that disrupts the hydrogen bonding network and the stability of membrane proteins via its interference with the intramolecular interactions mediated by non-covalent forces on cellular membranes, it was firstly shown that altering the membrane integrity as well as the native state of cellular membrane proteins, by weakening the hydrophobic effect, could attenuate the possible interaction of [omim][BF 4 ] with cellular membranes and the concomitant induction of protein-based intracellular processes, commonly linked with the induction of severe cellular damage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The possible emergence of praziquantel (PZQ)-tolerant and/or -resistant schistosomes requires the study and development of new antischistosomal drugs as alternatives to PZQ. The present study investigates the capability of 3 dyes-methylene blue (MB), neutral red (NR), and trypan blue (TB)-to assess the in vitro antischistosomal effect of antimalarial drugs on Schistosoma mansoni adult worms. S. mansoni adult worms were incubated in the medium alone as the control or in the medium supplemented with 10 μg/ml primaquine (PQ), artesunate (AR), or amodiaquine (AQ) for 5 days. Viabilities of the worms were observed following staining with MB, NR, or TB. The disparity of MB and NR staining among male and female adult worms treated with PQ, AR, and AQ correlated with the various levels of damage to the male and female worms. Furthermore, the severity of the damage to the adult worms treated with the 3 drugs appeared to be reflected in the TB staining status. The results indicate that the 3 non-fluorescent dyes can serve as useful complementary tools to assess the antischistosomal effect of antimalarial drugs.

Aflatoxins can be produced by 21 species within sections Flavi (16 species), Ochraceorosei (2), and Nidulantes (3) of the fungal genus Aspergillus. They pose risks to human and animal health due to high toxicity and carcinogenicity. Detecting aflatoxin producers can help to assess toxicological risks associated with contaminated commodities. Species specific molecular assays (PCR and LAMP) are available for detection of major producers, but fail to detect species of minor importance. To enable rapid and sensitive detection of several aflatoxin producing species in a single analysis, a nor1 gene-specific LAMP assay was developed. Specificity testing showed that among 128 fungal species from 28 genera, 15 aflatoxigenic species in section Flavi were detected, including synonyms of A. flavus and A. parasiticus. No cross reactions were found with other tested species. The detection limit of the assay was 9.03pg of A. parasiticus genomic DNA per reaction. Visual detection of positive LAMP reactions under daylight conditions was facilitated using neutral red to allow unambiguous distinction between positive and negative assay results. Application of the assay to the detection of A. parasiticus conidia revealed a detection limit of 211 conidia per reaction after minimal sample preparation. The usefulness of the assay was demonstrated in the analysis of aflatoxinogenic species in samples of rice, nuts, raisins, dried figs, as well as powdered spices. Comparison of LAMP results with presence/absence of aflatoxins and aflatoxin producing fungi in 50 rice samples showed good correlation between these parameters. Our study suggests that the developed LAMP assay is a rapid, sensitive and user-friendly tool for surveillance and quality control in our food industry., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study was designed to study the chemical composition of Cyclocarya paliurus polysaccharide and inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage. A new elution (0.3% NaCl aqueous solution) of Cyclocarya paliurus polysaccharide (CPP-3) was characterized by different methods such as fourier transform infrared spectra (FT-IR), UV-vis, gas chromatography-mass spectrometry (GC-MS), high performance gel chromatography (HPGLC) and scanning electron microscopy (SEM). Cell viability was measured by MTT test, phagocytosis assay was measured by Neutral red uptake assay, nitrite was measured by Griess assay, TNF-α and IL-1β analysis were measured by ELISA, PGE 2 was measured by enzyme immunoassay system. The results showed that CPP-3 was comprised of two polysaccharides with average molecular weight (Mw) of 5.69×10 4 Da and 4.94×10 3 Da. CPP-3 contains six monosaccharides, of which are rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), glucose (Glu), galactose (Gal), the molar ratio of six monosaccharides is 0.060:0.109:0.053:0.128:0.293:0.357. CPP-3 increased the amount of NO released from mouse macrophage RAW264.7 and significantly increased the levels of TNF-α, IL-1β and PGE 2 (P<0.01). CPP-3 suppressed LPS-stimulated RAW264.7 macrophage to release NO, TNF-α, IL-1β and PGE 2 (P<0.01). CPP-3 and LPS accounted for synergistic effect on the release of NO and TNF-α, CPP-3 and LPS accounted for antagonistic effect on the release of IL-1β and PGE 2 ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A polysaccharide (FVSP) was isolated from the base of Flammuliana Velutipes stipe, and FVSP was further purified by DEAE-cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography to obtain three fractions named FVSP-1, FVSP-2 and FVSP-3. Then their activation of macrophage cell RAW 264.7 and anti-proliferative effects to the murine melanoma B16F10 and fibroblasts L929 cells were evaluated by using the cell model experiments. The results indicated that the polysaccharide fractions could increase the proliferation and phagocytic activity of macrophage significantly and play an inhibited effect on the cancer cells. Moreover, the anti-proliferative activities of FVSPs increased with the participation of the antitumor factors induced from macrophage by polysaccharides fractions. Taken together, these results indicated that three polysaccharides fractions from the base of F. Velutipes stipe may be useful as potent antitumor agents for the prevention of tumorigenesis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Conversion of C1 gas feedstock, including carbon monoxide (CO), into useful platform chemicals has attracted considerable interest in industrial biotechnology. Nevertheless, the low conversion yield and/or growth rate of CO-utilizing microbes make it difficult to develop a C1 gas biorefinery process. The Wood-Ljungdahl pathway which utilize CO is a pathway suffered from insufficient electron supply, in which the conversion can be increased further when an additional electron source like carbohydrate or hydrogen is provided. In this study, electrode-based electron transference using a bioelectrochemical system (BES) was examined to compensate for the insufficient reducing equivalent and increase the production of volatile fatty acids. The BES including neutral red (BES-NR), which facilitated electron transfer between bacteria and electrode, was compared with BES without neutral red and open circuit control. The coulombic efficiency based on the current input to the system and the electrons recovered into VFAs, was significantly higher in BES-NR than the control. These results suggest that the carbon electrode provides a platform to regulate the redox balance for improving the bioconversion of CO, and amending the conventional C1 gas fermentation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep "saddles" that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1 H}- 15 N nuclear Overhauser effect, T 1 , and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.

Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is described. Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10® system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses., Goals of This Study: 1) demonstrate functionality of the VITROCELL® VC10® system by installation, operational and performance qualification, 2) develop and validate a cellular system for assessing cytotoxicity following whole smoke exposure and 3) assess the whole smoke NRU assay sensitivity for statistical differentiation between a reference combustible cigarette (3R4F) and a primarily "heat-not-burn" cigarette (Eclipse)., Results: The VITROCELL® VC10® provided consistent generation and delivery of whole smoke; exposure-related changes in in vitro cytotoxicity were observed with reproducible IC 50 values; comparative analysis showed that the heat-not-burn cigarette was significantly (P<0.001) less cytotoxic than the 3R4F combustible cigarette, consistent with the lower levels of chemical constituents liberated by primarily-heating the cigarette versus burning., (Copyright © 2017. Published by Elsevier Ltd.)

We investigated the use of one- and two-mediator systems in amperometric BOD biosensors (BOD, biochemical oxygen demand) based on the yeast Debaryomyces hansenii. Screening of nine mediators potentially capable of electron transfer - ferrocene, 1,1'-dimethylferrocene, ferrocenecarboxaldehyde, ferroceneacetonitrile, neutral red, 2,6-dichlorophenolindophenol, thionine, methylene blue and potassium ferricyanide - showed only ferrocene and neutral red to be efficient electron carriers for the eukaryotes studied. Two-mediator systems based on combinations of the investigated compounds were used to increase the efficiency of electron transfer. The developed two-mediator biosensors exceeded their one-mediator analogs by their characteristics. The most preferable two-mediator system for developing a BOD biosensor was a ferrocene-methylene blue combination that ensured a satisfactory long-time stability (43 days), selectivity, sensitivity (the lower limit of the determined BOD 5 concentrations, 2.5mg О 2 /dm 3 ) and speed (assay time for one sample, not greater than 10min) of BOD determination. Analysis of water samples showed that the use of a ferrocene-methylene blue two-mediator system and the yeast D. hansenii enabled registration of data that highly correlated with the results of the standard method (R=0.9913)., (Copyright © 2017 Elsevier Inc. All rights reserved.)

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC 50 ] = 0.03 μM) and Semliki Forest virus (SFV; EC 50 = 0.11 μM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate., (Copyright © 2017 American Society for Microbiology.)

A comparative study of binding interaction between Safranin O (SO) and Neutral Red (NR) with lysozyme (Lyz) has been reported using several spectroscopic methods along with computational approaches. Steady-state fluorescence measurements revealed static quenching as the major quenching mechanism in Lyz-SO and Lyz-NR interaction, which is further supported by time-resolved fluorescence and UV-vis measurements. Additionally, binding and thermodynamic parameters of these interactions are calculated from temperature dependent fluorescence data. Moreover, conformational changes of protein upon binding with SO and NR are provided by synchronous and circular dichroism (CD) measurements. Molecular docking study provided the exact binding location of SO and NR in lysozyme. Along with this study, molecular dynamics simulation is carried out to measure the stability of Lyz, Lyz-SO, and Lyz-NR complex. The present study revealed the strong binding affinity of dyes with lysozyme, and this study would be helpful toward medical and environmental science.

Brominated Tetra-BDE (BDE-47), is suggested to be widely distributed in marine environments and highly accumulated in marine organisms. Blue mussel Mytilus edulis is a sentinel organism that is commonly used for monitoring chemical contaminants in coastal ecosystems, and its haemocytes play an essential role in immune function. Therefore, we estimated the effects of BDE-47 exposure on the M. edulis haemocytes' immune function under controlled laboratory conditions. The study found the following results: (1) BDE-47 exposure increased the mortality of the haemocytes and decreased the total haemocyte counts. The ultrastructure and microstructure in the haemocytes were significantly changed, and the micronucleus frequency was increased steadily in a concentration-dependent manner, inferring that cellular and molecular damages occur during the exposure. (2) The immune function of the haemocytes was estimated from lysosomal and phagocytic changes. The lysosomal membrane stability was significantly disrupted compared to the control according to neutral red retention time changes, and the phagocytic ability was reduced significantly. Two lysosomal enzymes, acid phosphatases and alkaline phosphatases, presented similar increasing trends during the treatment. (3) BDE-47 exposure significantly induced the overproduction of reactive oxygen species and malondialdehyde in a clear time- and concentration-dependent manner, suggesting the occurrence of oxidative stress. We thus presumed that BDE-47 exposure affected the immune function of the mussel's haemocytes, and an ROS-mediated pathway might be one of the possible explanations for the observation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

The 3T3 neutral red uptake phototoxicity test (OECD TG432) is an alternative phototoxicity test method that is relatively easy and rapid to implement, with results obtainable in a short time, and is reported to have high reproducibility compared with in vivo assay methods. However, this method has been shown to be unsuitable for testing poorly water-soluble substances, which tend to separate out when mixed with the assay buffer solution. This causes difficulties in determining the dose dependency of substances and subsequent determination of the photoirritation factor because the ratio of cell viability, expressed as the half-maximal inhibitory concentration (IC 50 ) in the presence or absence of light, is not calculable. In this study, we investigated the optimum conditions for the evaluation of poorly water-soluble substances. In the conventional method, the final solvent concentration was 1% and the pre-incubation time was 60 min, but in the modified method, 10% and 5 min were used, respectively. Next, the results from the conventional method were compared with those of our modified method, which was found to be viable and comparable with the conventional method. Moreover, the false positive results frequently obtained with poorly water-soluble substances in the conventional method were not evident with the modified method, thus confirming its usefulness for the evaluation of such substances. We therefore propose that the modified method can be used for the in vitro testing of poorly water-soluble substances in phototoxicity evaluations.

We have synthesized two new complexes of platinum (1) and ruthenium (2) with α-amino acid, l-alanine, and 2,3-dihydroxybenzaldehyde derived Schiff base (L). The ligand and both complexes were characterized by using elemental analysis and several other spectroscopic techniques viz; IR, (1)H, (13)C NMR, EPR, and ESI-MS. Furthermore, the protein-binding ability of synthesized complexes was monitored by UV-visible, fluorescence and circular dichroism techniques with a model protein, human serum albumin (HSA). Both the PtL2 and RuL2 complexes displayed significant binding towards HSA. Also, in vitro cytotoxicity assay for both complexes was carried out on human hepatocellular carcinoma cancer (HepG2) cell line. The results showed concentration-dependent inhibition of cell viability. Moreover, the generation of reactive oxygen species was also evaluated, and results exhibited substantial role in cytotoxicity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Several testing methods have been established to identify potential phototoxins. The present study was undertaken to clarify the predictive ability of in vitro photosafety assays for photoallergenicity. On the basis of animal and/or clinical photosafety information, 23 photoallergens and 7 non-phototoxic/non-photoallergenic chemicals were selected and subjected to UV/VIS spectral analysis, reactive oxygen species (ROS)/micellar ROS (mROS) assays, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Of the photoallergens tested, ca. 96% of chemicals had intense UV/VIS absorption with a molar extinction coefficient of over 1000 M(-1) cm(-1), and false-positive predictions were made for 3 non-photoallergenic chemicals. In the ROS assay, all photoallergens were found to be potent ROS generators under exposure to simulated sunlight. In the photosafety prediction based on the ROS assay, the individual specificity was 85.7%, and the positive predictivity and negative predictivity were found to be 95.8% and 100%, respectively. Most of the photoirritant chemicals were correctly identified by the 3T3 NRU PT; however, it provided false predictions for ca. 48% of photoallergens. The orders of sensitivity and specificity for photoallergenicity prediction were estimated to be: [sensitivity] ROS assay>UV/VIS absorption ≫ 3T3 NRU PT, and [specificity] 3T3 NRU PT>ROS assay ≫ UV/VIS absorption. Thus, photochemical assays, in particular the ROS assay, can be used for assessment of photoallergenicity, although there were some false-positive predictions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

The aim of this work was to compare the effects of electrosynthesis on different bacterial species. The effects of neutral red-mediated electrosynthesis on the metabolite profiles of three microorganisms: Escherichia coli, Klebsiella pneumoniae, and Zymomonas mobilis, were measured and compared and contrasted. A statistically comprehensive analysis of neutral red-mediated electrosynthesis is presented using the analysis of end-product profiles, current delivered, and changes in cellular protein expression. K. pneumoniae displayed the most dramatic response to electrosynthesis of the three bacteria, producing 93% more ethanol and 76% more lactate vs. control fermentation with no neutral red and no electron delivery. Z. mobilis showed no response to electrosynthesis except elevated acetate titers. Stoichiometric comparison showed that NAD(+) reduction by neutral red could not account for changes in metabolites during electrosynthesis. Neutral red-mediated electrosynthesis was shown to have multifarious effects on the three species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HCS) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The neutral red retention time (NRRT) assay is useful for detecting decreased lysosomal membrane stability in haemocytes sampled from bivalves, a phenomenon often associated with exposure to environmental pollutants including nanomaterials. Bivalves are popular sentinel species in ecotoxicology and use of NRRT in study of species in the genus Mytilus is widespread in environmental monitoring. The NRRT assay has been used as an in vivo test for toxicity of carbon nanoparticles (Moore MN, Readman JAJ, Readman JW, Lowe DM, Frickers PE, Beesley A. 2009. Lysosomal cytotoxicity of carbon nanoparticles in cells of the molluscan immune system: An in vivo study. Nanotoxicology. 3 (1), 40-45). We here report application of this assay adapted to a microtitre plate format to a panel of metal and metal oxide nanoparticles (2 ppm). This showed that copper, chromium and cobalt nanoparticles are toxic by this criterion while gold and titanium nanoparticles are not. As the former three nanoparticles are often reported to be cytotoxic while the latter two are thought to be non-cytotoxic, these data support use of NRRT as a general in vitro assay in nanotoxicology., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In vitro toxicological studies for tobacco product assessment have traditionally been undertaken using the particulate phase of tobacco smoke. However, this does not truly reflect exposure conditions that occur in smokers. Thus in vitro cell culture systems are required in which cells are exposed to tobacco whole smoke (WS) at the air-liquid interface (ALI). In this study bronchial epithelial cells were cultured on semi-permeable membranes, transitioned to the ALI and the robustness and sensitivity of the cells to tobacco WS and vapour phase (VP) assessed. Although no effect of air exposure was observed on cell viability, IL-6 and IL-8 release was increased. Exposure to WS resulted in a significant dose dependent decrease in cell viability and a significant non-dose dependent increase in inflammatory mediator secretion. The VP was found to contribute approximately 90% of the total cytotoxicity derived from WS. The cell culture system was also able to differentiate between two smoking regimens and was sensitive to passage number with increased inflammatory mediator secretion and lower cell viability observed in cell cultures of low passage number following WS exposure. This simple cell culture system may facilitate studies on the toxicological impact of future tobacco products and nicotine delivery devices., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this work was to elucidate the mechanism of mediated microbial electrosynthesis via neutral red from an electrode to fermenting Escherichia coli cultures in a bioelectrochemical system. Chemical reduction of NAD(+) by reduced neutral red did not occur as predicted. Instead, neutral red was shown to reduce the menaquinone pool in the inner bacterial membrane. The reduced menaquinone pool altered fermentative metabolite production via the arcB redox-sensing cascade in the absence of terminal electron acceptors. When the acceptors DMSO, fumarate, or nitrate were provided, as many as 19% of the electrons trapped in the reduced acceptors were derived from the electrode. These results demonstrate the mechanism of neutral red-mediated microbial electrosynthesis during fermentation as well as how neutral red enables microbial electrosynthesis of reduced terminal electron acceptors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nickel ferrite nanoparticles (NPs) have received much attention for their potential applications in biomedical fields such as magnetic resonance imaging, drug delivery and cancer hyperthermia. However, little is known about the toxicity of nickel ferrite NPs at the cellular and molecular levels. In this study, we investigated the cytotoxic responses of nickel ferrite NPs in two different types of human cells (i.e., liver HepG2 and breast MCF-7). Nickel ferrite NPs induced dose-dependent cytotoxicity in both types of cells, which was demonstrated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) assays. Nickel ferrite NPs were also found to induce oxidative stress, which was evident by the depletion of glutathione and the induction of reactive oxygen species (ROS) and lipid peroxidation. The mitochondrial membrane potential due to nickel ferrite NP exposure was also observed. The mRNA levels for the tumor suppressor gene p53 and the apoptotic genes bax, CASP3 and CASP9 were up-regulated, while the anti-apoptotic gene bcl-2 was down-regulated following nickel ferrite NP exposure. Furthermore, the activities of apoptotic enzymes (caspase-3 and caspase-9) were also higher in both types of cells treated with nickel ferrite NPs. Cytotoxicity induced by nickel ferrite was efficiently prevented by N-acetyl cysteine (ROS scavenger) treatment, which suggested that oxidative stress might be one of the possible mechanisms of nickel ferrite NP toxicity. We also observed that MCF-7 cells were slightly more susceptible to nickel ferrite NP exposure than HepG2 cells. This study warrants further investigation to explore the potential mechanisms of different cytotoxic responses of nickel ferrite NPs in different cell lines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Previously, a non-animal screening approach was proposed for evaluating photosafety of cosmetic ingredients by means of in vitro photochemical and photobiochemical assays; however, complex cosmetic ingredients, such as plant extracts and polymers, could not be evaluated because their molecular weight is often poorly defined and so their molar concentration cannot be calculated. The aim of the present investigation was to establish a photosafety screen for complex cosmetic ingredients by using appropriately modified in vitro photosafety assays. Twenty plant extracts were selected as model materials on the basis of photosafety information, and their phototoxic potentials were assessed by means of ultraviolet (UV)/visible light (VIS) spectral analysis, reactive oxygen species (ROS)/micellar ROS (mROS) assays, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). The maximum UV/VIS absorption value was employed as a judgment factor for evaluating photoexcitability of samples, and the value of 1.0 was adopted as a tentative criterion for photosafety identification. The ROS/mROS assays were conducted at 50 μg/mL, and no false negative prediction was obtained. Furthermore, the ROS/mROS assays at 50 μg/mL had a similar predictive capacity to the ROS/mROS assays in the previous study. A systematic tiered approach for simple and rapid non-animal photosafety evaluation of complex cosmetic ingredients can be constructed using these modified in vitro photochemical assays., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Coelomocytes comprise the immune system of earthworms and due to their sensitivity responding to a wide range of pollutants have been widely used as target cells in soil ecotoxicology. Recently, in vitro assays with primary cultures of coelomocytes based in the neutral red uptake (NRU) assay have been developed as promising tools for toxicity assessment chemical in a reproducible and cost-effective manner. However, NRU showed a bimodal dose-response curve previously described after in vivo and in vitro exposure of earthworm coelomocytes to pollutants. This response could be related with alterations in the relative proportion of coelomocyte subpopulations, amoebocytes and eleocytes. Thus, the aims of the present work were, first, to establish the toxicity thresholds that could be governed by different cell-specific sensitivities of coelomocytes subpopulations against a series of metals (Cu, Cd, Pb, Ni), and second to understand the implication that coelomocyte population dynamics (eleocytes vs. amoebocytes) after exposure to pollutants can have on the viability of coelomocytes (measured by NRU assay) as biomarker of general stress in soil health assessment. Complementarily flow cytometric analyses were applied to obtain correlative information about single cells (amoebocytes and eleocytes) in terms of size and complexity, changes in their relative proportion and mortality rates. The results indicated a clear difference in sensitivity of eleocytes and amoebocytes against metal exposure, being eleocytes more sensitive. The bimodal dose-response curve of NRU after in vitro exposure of primary cultures of coelomocytes to metals revealed an initial mortality of eleocytes (decreased NRU), followed by an increased complexity of amoebocytes (enhanced phagocytosis) and massive mortality of eleocytes (increased NRU), to give raise to a massive mortality of amoebocytes (decrease NRU). A synergistic effect on NRU was exerted by the exposure to high Cu concentrations and acidic pH (elicited by the metal itself), whereas the effects on NRU produced after exposure to Cd, Ni and Pb were due solely to the presence of metals, being the acidification of culture medium meaningless.

Tobacco smoke from a combustible cigarette contains more than 6000 constituents; approximately 150 of these are identified as toxicants. Technologies that modify the tobacco blend to reduce toxicant emissions have been developed. These include tobacco sheet substitute to dilute toxicants in smoke and blend treated tobacco to reduce the levels of nitrogenous precursors and some polyphenols. Filter additives to reduce gas (vapour) phase constituents have also been developed. In this study, both tobacco blend and filter technologies were combined into an experimental cigarette and smoked to International Organisation on Standardisation and Health Canada puffing parameters. The resulting particulate matter was subjected to a battery of in vitro genotoxicity and cytotoxicity assays - the Ames test, mouse lymphoma assay, the in vitro micronucleus test and the Neutral Red Uptake assay. The results indicate that cigarettes containing toxicant reducing technologies may be developed without observing new additional genotoxic hazards as assessed by the assays specified. In addition, reductions in bacterial mutagenicity and mammalian genotoxicity of the experimental cigarette were observed relative to the control cigarettes. There were no significant differences in cytotoxicity relative to the control cigarettes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated., Results: Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC50), while IC50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays., Conclusions: This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.

This study aimed to assess the phototoxic potential of combined UV-filters and retinyl palmitate (RP) in the presence or not of bemotrizinol (BMTZ), employing photostability and in vitro and in vivo phototoxicity assays. The formulations tested contained octocrylene (OCT), octyl methoxycinnamate (OMC), benzophenone-3 (BZP-3) and RP (photostable) or octocrylene (OCT), octyl methoxycinnamate (OMC), avobenzone (AVO) and RP (less photostable). Both formulations were supplemented with bemotrizinol. Photostability was evaluated by exposing, or not, formulations spread on a glass plate to UVA/UVB irradiation. The resulting products were quantified by HPLC analysis. In vitro phototoxicity of UV-filters and combinations were evaluated using 3T3 viable monolayer fibroblast cultures submitted, or not, to irradiation according to OECD TG 432. In vivo photoallergy and photoxicity were assessed by clinical studies (photopatch test). Photostability assays showed that UV-filter bemotrizinol was a better photostabilizer for RP/benzophenone-3 than for RP/avobenzone. The in vitro phototoxicity of the combination RP/avobenzone was reduced by bemotrizinol. Clinical studies did not indicate phototoxic or photoallergenic potentials in all formulations tested. It is concluded that the 3T3 NRU phototoxicity test may be considered a supplementary assay in formulation developments, since it can detect chemically unstable and potentially phototoxic combinations. However, extrapolation of in vitro positive results to human photopatch tests may be performed only to a limited extent., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Tobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity., Findings: The findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference., Conclusions: We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.

We report herein the antitumor actions of three oxidovanadium(IV) complexes on MG-63 human osteosarcoma cell line. The three complexes: VO(oda), VO(oda)bipy and VO(oda)phen (oda=oxodiacetate), caused a concentration dependent inhibition of cell viability. The antiproliferative action of VO(oda)phen could be observed in the whole range of concentrations (at 2.5 μM), while VO(oda)bipy and VO(oda) showed a decrease of cell viability only at higher concentrations (at 50 and 75 μM, respectively) (p<0.01). Moreover, VO(oda)phen caused a decrease of lysosomal and mitochondrial activities at 2.5 μM, while VO(oda) and VO(oda)bipy affected neutral red uptake and mitochondrial metabolism at 50 μM (p<0.01). On the other hand, no DNA damage studied by the Comet assay could be observed in MG-63 cells treated with VO(oda) at 2.5-10 μM. Nevertheless, VO(oda)phen and VO(oda)bipy induced DNA damage at 2.5 and 10 μM, respectively (p<0.01). The generation of reactive oxygen species increased at 10 μM of VO(oda)phen and only at 100 μM of VO(oda) and VO(oda)bipy (p<0.01). Besides, VO(oda)phen and VO(oda)bipy triggered apoptosis as determined by externalization of the phosphatidylserine. The determination of DNA cleavage by agarose gel electrophoresis showed that the ability of VO(oda)(bipy) is similar to that of VO(oda), while VO(oda)(phen) showed the highest nuclease activity in this series. Overall, our results showed a good relationship between the bioactivity of the complexes and their structures since VO(oda)phen presented the most potent antitumor action in human osteosarcoma cells followed by VO(oda)bipy and then by VO(oda) according to the number of intercalating heterocyclic moieties., (© 2013.)

The immunotoxic effects of heavy metals on blood leukocytes of sea bass (Dicentrarchus labrax) were examined. The cells, separated by a discontinuous Percoll-gradients, were exposed in vitro to various sublethal concentrations of cadmium and copper (10(-7)M, 10(-5)M, and 10(-3)M) and their immunotoxic effect was then evaluated by measuring neutral red uptake, MTT assay, DNA fragmentation and Hsp70 gene expression. First of all, we demonstrated that the cells treated in vitro could incorporate Cd and Cu. A relationship between heavy metal exposure and dose-time-dependent alterations in responses of leukocytes from blood was found for both metals, but copper was more immunotoxic than cadmium in all assays performed. A significant reduction in the cells׳ ability to uptake neutral red and viability by MTT assay was recorded, indicating that both cadmium and copper could change the membrane permeability, inducing cellular apoptosis when the concentration of metals reached 10(-3)M. The apoptotic effect may also explain the high level of cytotoxicity found when the leukocytes were exposed to higher concentration of metals. These results demonstrated that toxic effect of copper and cadmium affect on the mechanisms of cell-mediated immunity reducing the immune defences of the organism., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Given that polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PH), possess a potent risk for aquatic biota, a great attempt to develop and apply advanced oxidation processes, such as ultrasound (US), is of great concern nowadays. However, because US PAH-derived toxic intermediates are difficult to detect, the present study investigates aqueous PH toxicity before and after high-frequency US degradation, in hemocytes of mussel Mytilus galloprovincialis. Specifically, cell viability (with the use of neutral red uptake/NRU method), and oxidative-stress indices in terms of superoxide anions, (O2(-)), nitric oxides (NO, in terms of nitrites), lipid peroxidation products (in terms of malondialdehyde/MDA content) and DNA damage (with the use of Comet assay method) were investigated in mussel hemocytes exposed to environmentally relevant concentrations of PH (0.01, 0.1, 1 and 10 μg L(-1)), before and after US treatment for 120 min (at a frequency of 582 kHz). According to the results, the NRU method showed a significant attenuation of PH-induced mortality in US PH-treated hemocytes in all cases. Moreover, the increased levels of O2(-) and NO generation, as well as MDA content measured in PH-treated hemocytes, were drastically decreased after US degradation in any case. Similarly, the disturbance of DNA integrity (in terms of % DNA in tail, OM and TM), was negligible in case of US PH-treated hemocytes. Although further in vitro and in vivo studies are needed, the present study showed for the first time that high frequency US could be applied as a highly efficient and "environmentally friendly" process for degrading low molecular weight PAH, such as PH., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Perfluorinated chemicals (PFCs) are extremely persistent and have been found extensively in the environment and wildlife. Oceans are the final sink for many persistent organic pollutants (POPs) including PFCs. However, to date, there has been a lack of studies that investigated the environmental consequences of PFCs on marine organisms. To fill in this gap, environmental toxicity of two dominant PFCs, perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), was examined in a sentinel species, green mussel Perna viridis, using a series of biomarkers corresponding to different biological levels (molecular, cellular, and physiological). Correlations among these biomarkers were also investigated. The results showed that the tested compounds can induce a series adverse effect at different biological levels, including oxidative stress, DNA damage, membrane instability, suppressed filtration rate, and reduced body weight. Correlation analysis revealed that excess production of reactive oxygen species could be the major toxic pathway. An indirect mode of toxic action was also explored where adverse impacts could be secondary effects of PFC exposure. The joint analysis of biomarkers from multiple biological levels resulted in a comprehensive understanding of how PFC exposure can influence the health of organisms. The correlations of these biomarkers also provided a new perspective of the ecological consequences of PFCs.

Phototoxicity is a relatively common phenomenon and is an adverse effect of some systemic drugs. The fundamental initial step of photochemical reactivity is absorption of a photon; however, little guidance has been provided thus far regarding how ultraviolet-visible (UV-vis) light absorption spectra may be used to inform testing strategies for investigational drugs. Here we report the results of an inter-laboratory study comparing the data from harmonized UV-vis light absorption spectra obtained in methanol with data from the in vitro 3T3 Neutral Red Uptake Phototoxicity Test. Six pharmaceutical companies submitted data according to predefined quality criteria for 76 compounds covering a wide range of chemical classes showing a diverse but "positive"-enhanced distribution of photo irritation factors (22%: PIF<2, 12%: PIF 2-5, 66%: PIF>5). For compounds being formally positive (PIF value above 5) the lowest reported molar extinction coefficient (MEC) was 1700 L mol⁻¹ cm⁻¹ in methanol. However, the majority of these formally positive compounds showed MEC values being significantly higher (up to almost 40,000 L mol⁻¹ cm⁻¹). In conclusion, an MEC value of 1000 L mol⁻¹ cm⁻¹ may represent a reasonable and pragmatic threshold warranting further experimental photosafety evaluation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Several studies have suggested that silicon (Si) may be essential for the normal development of connective tissue and the skeleton. Positive effects of Si from the diet as well as from Si-containing biomaterials, such as bioactive glass 45S5 (BG), have been demonstrated. Studies have reported that Si stimulates osteoblast proliferation and differentiation. However, the effects of Si on osteoclasts have not been directly addressed. The purpose of the present in vitro study was to clarify if Si has regulatory effects on osteoclast formation and bone resorption. The effects of BG, BG dissolution extracts and Si containing cell culture medium were investigated in a mouse calvarial bone resorption assay and osteoclast formation assays (mouse bone marrow cultures and RAW264.7 cell cultures). We conclude from our results that Si causes significant inhibition of osteoclast phenotypic gene expressions, osteoclast formation and bone resorption in vitro. In conclusion, the present study suggests that Si has a dual nature in bone metabolism with stimulatory effects on osteoblasts and inhibitory effects on osteoclasts. This suggested property of Si might be interesting to further explore in future biomaterials for treatments of bone defects in patients., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The main purpose of the present study was to establish a non-animal photosafety assessment approach for cosmetics using in vitro photochemical and photobiochemical screening systems. Fifty-one cosmetics, pharmaceutics and other chemicals were selected as model chemicals on the basis of animal and/or clinical photosafety information. The model chemicals were assessed in terms of photochemical properties by UV/VIS spectral analysis, reactive oxygen species (ROS) assay and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Most phototoxins exhibited potent UV/VIS absorption with molar extinction coefficients of over 1000M(-1)cm(-1), although false-negative prediction occurred for 2 cosmetic phototoxins owing to weak UV/VIS absorption. Among all the cosmetic ingredients, ca. 42% of tested chemicals were non-testable in the ROS assay because of low water solubility; thereby, micellar ROS (mROS) assay using a solubilizing surfactant was employed for follow-up screening. Upon combination use of ROS and mROS assays, the individual specificity was 88.2%, and the positive and negative predictivities were estimated to be 94.4% and 100%, respectively. In the 3T3 NRU PT, 3 cosmetics and 4 drugs were incorrectly predicted not to be phototoxic, although some of them were typical photoallergens. Thus, these in vitro screening systems individually provide false predictions; however, a systematic tiered approach using these assays could provide reliable photosafety assessment without any false-negatives. The combined use of in vitro assays might enable simple and fast non-animal photosafety evaluation of cosmetic ingredients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Alpinia pahangensis, a wild ginger distributed in the lowlands of Pahang, Malaysia, is used by the locals to treat flatulence. In this study, the antioxidant and cytotoxic activities of the crude aqueous methanol and fractionated extracts of Alpinia pahangensis against five different cancer and one normal cell lines were investigated. The total phenolic content of each extract and its fractions were also quantified. This is the first report on the antioxidant and cytotoxic activities of Alpinia pahangensis extract., Methods: In the current study, the crude methanol and fractionated extract of the rhizomes of Alpinia pahangensis were investigated for their antioxidant activity using four different assays namely, the DPPH scavenging activity, superoxide anion scavenging, β-carotene bleaching and reducing power assays whilst their phenolic contents were measured by the Folin-Ciocalteu's method.In vitro neutral red cytotoxicity assay was employed to evaluate the cytotoxic activity against five different cancer cell lines, colon cancer (HCT 116 and HT-29), cervical cancer (Ca Ski), breast cancer (MCF7) and lung cancer (A549) cell lines, and one normal cell line (MRC-5). The extract that showed high cytotoxic activity was further investigated for its chemical constituents by GC-MS (gas chromatography-mass spectrometry) analysis., Results: The ethyl acetate fraction showed the strongest DPPH radical scavenging (0.35 ± 0.094 mg/ml) and SOD activities (51.77 ± 4.9%) whilst the methanol extract showed the highest reducing power and also the strongest antioxidant activity in the β-carotene bleaching assays in comparison to other fractions. The highest phenolic content was found in the ethyl acetate fraction, followed by the crude methanol extract, hexane and water fractions. The results showed a positive correlation between total phenolic content with DPPH radical scavenging capacities and SOD activities. The hexane fraction showed potent cytotoxic effect against KB, Ca Ski and HCT 116 cell lines with IC₅₀ of 5.8 ± 0.1 and 9.1 ± 2.0 ug/ml, respectively. The major components of hexane fraction analysed by GC-MS analysis were mostly methyl esters., Conclusions: The current study suggests that the methanol extract and ethyl acetate fraction of A. pahangensis is a potential source of natural antioxidant for protective as well as prevention of life-threatening diseases. The hexane fraction of A. pahangensis may have the potential to be developed into therapeutic option for treating cancer.