Reduction Of Tetrazolium Salt Research Articles

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium WST-1.

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric microbial viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7-14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3-4 days using the WST-1 colorimetric method and those obtained after 10-14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.

On the photocatalytic reduction of MTT tetrazolium salt on the surface of TiO2 nanoparticles: Formazan production kinetics and mechanism

HypothesisThe MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] cell cytotoxicity indicator is photocatalytically reduced on the surface of TiO2 nanoparticles in phosphate-buffered-saline (PBS) environment. We hypothesize that specific phosphate adsorption may be used to modulate the efficiency of the TiO2–MTT reaction through colloidal and semiconductor-liquid interface processes. ExperimentsThe TiO2–MTT reaction kinetics was studied in PBS, with respect to photocatalyst and MTT concentrations and irradiation wavelength. The effects of PBS and electron scavengers (Fe3+ ions) on reaction efficiency and the role of colloidal surface charge in the photocatalytic process were investigated. The structural and spectroscopic characteristics of relevant TiO2-formazan systems were studied by X-ray diffraction, transmission electron microscopy and IR-spectroscopy. FindingsThe reaction was pseudo-first order with respect to photocatalyst and showed a negative and fractional partial order with respect to MTT. Formazan production rates were directly proportional to radiation wavelength and TiO2 concentration and inversely proportional to the MTT initial concentration. The addition of Fe3+ ions, as well as the absence of PBS, induced strong reaction inhibition. Reaction efficiency and catalyst Zeta potential were enhanced by Na2HPO4 (PBS component) and showed a maximum around the phosphate concentration 0.005M.Structural/spectroscopic characterization confirmed the formation of amorphous MTT-formazan on the surface of TiO2 and the TiO2–phosphate binding.

Transcriptional profile of the human pathogenic fungus Paracoccidioides lutzii in response to sulfamethoxazole.

Paracoccidioidomycosis (PCM) is the most prevalent mycosis in Latin America and is caused by a group of fungi within the Paracoccidioides genus. The disease may present clinical and pathological manifestations ranging from asymptomatic pneumonia pulmonary lesions, to disseminated forms involving multiple organs. Sulfonamides were the first drugs used to treat PCM and are still used against this fungal infection. Sulfa drugs are competitive antagonists of ρ-aminobenzoic acid (PABA), a reaction catalyzed by dihydropteroate synthase (DHPS). However, the molecular effects of sulfonamides against the Paracoccidioides genus are unknown. The aim of this work was to investigate the global mechanism of action of sulfamethoxazole on Paracoccidioides lutzii. Yeast cells were grown on minimum medium in the presence or absence of sulfamethoxazole to construct EST libraries. The representational difference analysis (RDA) technique was used to identify up- and down-regulated P. lutzii genes after treatment with sulfamethoxazole. Approximately six transcripts related to mitochondrial function were differentially expressed. To confirm the RDA and bioinformatics results, several relevant genes were studied with quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate their levels of expression. To confirm the impact of sulfamethoxazole on mitochondria, we measured the reduction of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by P. lutzii with or without exposure to the drug. MTT assays reveal that sulfamethoxazole produces a marked dose-dependent adverse effect on P. lutzii. The transcriptional activity of selected genes in infected macrophages corroborated our in vitro results. The results indicated that sulfamethoxazole acts in P. lutzii as a competitor for amino acid, nucleic acids and folate cofactor biosynthesis, disrupting mitochondrial functions.

<i>In Vitro</i> Cytotoxicity and Mutagenicity Evaluation of Dental Leucite Glass-Ceramics from Local High Grade Silica Sand

The objective of the study was to determine the degree of biocompatibility of leucite glass-ceramics that have been produced from local high grade silica sand in terms of cytotoxicity and mutagenicity assays. In the present study, the cyctotoxicity and mutagenicity were studied using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay (MTT) and Ames Reverse Mutation. In the MTT assay, a dose response cytotoxicity of leucite sample was evaluated in L929 cells. The cells were treated with the concentrations of 6.25, 12.5, 25.0, 50.00, 100.00 and 200.00 mg/ml of the leucite sample for 24 hours. The cytotoxicity was determined by assessing the cell viability through the reduction of tetrazolium salts (MTT). The mutagenenicity of leucite sample was evaluated inS. typhiriumTA98. TA100, TA1535, TA1537 andE. coliWP2 in the Ames Reverse Mutation assay. Mutagenic effects were evaluated by comparing the mean number of revertant colonies of each extract concentraction with mean number of revertant colonies of the negative control. In results of MTT assay evaluated that the leucite did not show a cytotoxic effect at all concentrations under the condition of the study. Ames Reverse Mutation assay result proven that the leucite sample did not demonstrate a mutagenic effect under the condition of this study withSalmonella typhimuriumandEscherichia coli.

Influence of the addition of β-TCP on the morphology, thermal properties and cell viability of poly (lactic acid) fibers obtained by electrospinning.

Electrospinning is a simple and low-cost way to fabricate fibers. Among the various polymers used in electrospinning process, the poly (lactic acid) (PLA) stands out due to its excellent biodegradability and biocompatibility. Calcium phosphate ceramics has been recognized as an attractive biomaterial because their chemical composition is similar to the mineral component of the hard tissue in the body. Furthermore, they are bioactive and osteoinductive and some are even quite biodegradable. The beta-tricalcium phosphate (β-TCP) particles were synthesized by solid state reaction. Different contents of β-TCP particles were incorporated in polymer matrices to form fibers of PLA/β-TCP composites by electrospinning aiming a possible application as a scaffold for tissue engineering. The fibers were characterized by scanning electron microscopy (SEM), infrared (FTIR), differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA). The average diameter of the fibers varied in the range of 260-519.6 ± 50 nm. The presence of β-TCP particles promoted changes on thermal properties of the fibers. The composite with 8 wt-% of β-TCP showed a low degree of crystallinity and can be used for application in tissue engineering. The cell viability was analyzed by reduction of the methyl tetrazolium salt by the pyruvate dehydrogenase enzymatic complex present in the matrix of mitochondria (MTT test). All PLA fiber groups, with different contents of β-TCP, showed cytocompatibility ability with non-cytotoxicity effect and bioactive properties using SBF assay.

Cytotoxicity Screening of Modified Chitosan Derivatives Film on Primary Human Dermal Fibroblasts

Chitosan has been proposed for use in biomedical applications because of its biocompatibility and abundance in nature. However, many barriers to the use of chitosan in biomedical applications still exist due to its physical and chemical limitations. Therefore, the biosafety of this chitosan-derived product requires an in-vitro evaluation. The objective of this study is to establish a primary fibroblast culture from human skin tissue and to assess the cyto-compatibility of locally produced chitosan derivatives products. Primary human dermal fibroblasts were isolated by using enzymatic digestion techniques and characterized by immunocytochemical staining. Characterization has been done by using antibodies against heat shock protein 47 (HSP47) and fibroblasts surface protein (FSP) antibody confirmed that the cells were fibroblast. The cytotoxicity of the chitosan derivative products on fibroblast cell culture were compared by measuring cell survival as determined by the tetrazolium salt reduction assay. The results of cytotoxicity testing on fibroblasts using both direct and indirect contact approach showed that oligo chitosan and Ncarboxymethyl chitosan (N-CMC) were the most biocompatible biomaterials in this screening process. Therefore, both locally produced chitosan materials can be used as possible materials for biomedical applications.

Pulmonary Immune Responses to Aspergillus Fumigatus in Rats

ObjectiveTo evaluate immunologic mechanisms underlying Aspergillus fumigatus pulmonary infections in immunocompetent Dark Agouti (DA) and Albino Oxford (AO) rats recognized as being susceptible to some inflammatory diseases in different manners. MethodsLung fungal burden (quantitative colony forming units, CFU, assay), leukocyte infiltration (histology, cell composition) and their function (phagocytosis, oxidative activity, CD11b adhesion molecule expression) and cytokine interferon-γ (IFN-γ) and interleukin-17 and −4 (IL-17 and IL-4) lung content were evaluated following infection (intratracheally, 1×107 conidia). ResultsSlower reduction of fungal burden was observed in AO rats in comparison with that in DA rats, which was coincided with less intense histologically evident lung cell infiltration and leukocyte recovery as well as lower level of most of the their activities including intracellular myeloperoxidase activity, the capacity of nitroblue tetrazolium salt reduction and CD11b adhesion molecule expression (except for phagocytosis of conidia) in these rats. Differential patterns of changes in proinflammatory cytokine levels (unchanged levels of IFN-γ and transient increase of IL-17 in AO rats vs continuous increase of both cytokines in DA rats) and unchanged levels of IL-4 were observed. ConclusionGenetically-based differences in the pattern of antifungal lung leukocyte activities and cytokine milieu, associated with differential efficiency of fungal elimination might be useful in the future use of rat models in studies of pulmonary aspergillosis.

Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.

Ethnopharmacological relevanceLeaves and rhizomes of Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl. traditionally used in the Yanesha pharmacopoeia to treat skin infections such as leishmaniasis ulcers, or to reduce fever were chemically investigated to identify leishmanicidal compounds, as well as PPARγ activators. MethodsCompounds were isolated through a bioassay-guided fractionation and their structures were determined via detailed spectral analysis. The viability of Leishmania amazonensis axenic amastigotes was assessed by the reduction of tetrazolium salt (MTT), the cytotoxicity on macrophage was evaluated using trypan blue dye exclusion method, while the percentage of infected macrophages was determined microscopically in the intracellular macrophage-infected assay. The CD36, mannose receptor (MR) and dectin-1 mRNA expression on human monocytes-derived macrophages was evaluated by quantitative real-time PCR. ResultsSix sesquiterpenes (1–6), one dihydrobenzofuranone (7) and four flavonoids (8–11) were isolated from the leaves. Alongside, two flavonoids (12–13) and five diarylheptanoids (14–18) were identified in the rhizomes. Leishmanicidal activity against Leishmania amazonensis axenic amastigotes was evaluated for all compounds. Compounds 6, 7, and 11, isolated from the leaves, showed to be the most active derivatives. Diarylheptanoids 14–18 were also screened for their ability to activate PPARγ nuclear receptor in macrophages. Compounds 17 and 18 bearing a Michael acceptor moiety strongly increased the expression of PPARγ target genes such as CD36, Dectin-1 and mannose receptor (MR), thus revealing interesting immunomodulatory properties. ConclusionsPhytochemical investigation of Renealmia thyrsoidea has led to the isolation of leishmanicidal compounds from the leaves and potent PPARγ activators from the rhizomes. These results are in agreement with the traditional uses of the different parts of Renealmia thyrsoidea.

The effects of biogenic and abiogenic disulphides on endothelial cells in culture: Comparison of three methods of viability assessment

Effects of biogenic and abiogenic disulphides on viability of human umbilical vein endothelial cells in culture has been investigated using three methods: the neutral red uptake assay, quantification of intracellular ATP, and modifications of Mosmann method, the essence of which is the reduction of tetrazolium salts, MTT and MTS, by cells. 2,2'-dithio-bis(N,N-diethyl)ethanamine (DS) was used as an abiogenic disulphide. As for biogenic disulphides, we used GSSG and garlic oil (GO), the principal component of which is diallyl disulphide (DADS). It has been found that DS and GO have a similar cytotoxic effect upon the endothelial cells (EC50 - 0.6 mM). GSSG in concentrations up to 1 mM did not effect the viability of endothelial cells. It has been demonstrated for the first time that DS and GO can serve as mediators of plasma membrane oxidoreductase activity, tetrazolium salts being as the substrate; this may cause false-negative effect. Thus, the Mosmann method has serious limitations when testing the cytotoxicity of disulphides, though can be used in studying the mechanism of action of disulphides.

Molecular size and origin do not influence the harmful side effects of hydroxyethyl starch on human proximal tubule cells (HK-2) in vitro.

Recently, clinical trials revealed renal impairment induced by hydroxyethyl starch (HES) in septic patients. In prior studies, we managed to demonstrate that HES accumulated in renal proximal tubule cells (PTCs). The related pathomechanism has not yet been discovered. To validate our hypothesis that the HES molecule itself is harmful, regardless of its molecule size or origin, we conducted a comprehensive study to elucidate the influences of different HES preparations on PTC viability in vitro. Cell viability of human PTC was measured with a cytotoxicity assay, quantifying the reduction of tetrazolium salt to colored formazan. Experiments were performed by assessing the influence of different carrier solutions of HES (balanced, nonbalanced, culture medium), different average molecular weights (70, 130, 200 kDa), different origins (potato or corn derived), and various durations of incubation (2-21 hours). Furthermore, HES 130/0.4 was fractionated by ultrafiltration, and the impact on cell viability of average single-size fractions with <3, 3 to 10, 10 to 30, 30 to 50, 50 to 100, and >100 kDa was investigated. We also tested the possible synergistic effects of inflammation induced by tumor necrosis factor-α. All tested HES solutions, regardless of origin or carrier matrix, decreased cell viability in an equivalent, dose-dependent manner. Coincubation with tumor necrosis factor-α did not reduce HES-induced reduction of cell viability. Minor differences were detected comparing 70, 130, and 200 kDa preparations. Analysis of fractionated HES revealed that each fraction decreased cell viability. Even small HES molecules (10-30 kDa) were significantly deleterious. For the first time, we were able to show that only the total mass of HES molecules applied is responsible for the harmful impact on renal PTC in vitro. Neither molecular size nor their origin showed any relevance.

Differential suppressive effects of testosterone on immune function in fresh water snake, Natrix piscator: an in vitro study.

Reptiles represent the crucial phylogenetic group as they were the ancestors of both birds and mammals hence very important to study. The objectives of the present study were to investigate the potential roles of testosterone in the innate immune responses and splenic lymphocyte proliferation in fresh water snake, Natrix piscator. Animals were mildly anesthetized and spleens were taken out to study the splenic macrophage phagocytosis, super oxide production and nitrite release using in vitro testosterone. Splenic lymphocytes were isolated by density gradient centrifugation and were studied for mitogen induced proliferation in presence of in vitro testosterone. Testosterone suppressed the phagocytosis and nitrite release in a concentration dependent manner. Biphasic suppressive effect of testosterone was observed in superoxide production as judged by reduction of nitroblue tetrazolium salt where salt reduction was suppressed at lower and higher concentrations of testosterone. Mitogen induced splenic lymphocyte proliferation was also suppressed by testosterone. By suppressing immune responses, testosterone may, therefore, act as a physiological mechanism regulating the relative amount of energy invested into either reproductive effort or immunocompetence.

Glucose restriction decreases telomerase activity and enhances its inhibitor response on breast cancer cells: possible extra-telomerase role of BIBR 1532.

BackgroundConsiderable progress has been made to understand the association between lifestyle and diet in cancer initiation and promotion. Because excessive glucose consumption is a key metabolic hallmark of cancer cells, glucose restriction (GR) decreases the proliferation, and promotes the differentiation and transformation of cancer cells to quiescent cells. The immortality of cancerous cells is largely assured by telomerase, which is an interesting target for inhibition by BIBR 1532. In this study, we investigated the effect of GR on telomerase activity and on the efficacy of its inhibition by BIBR 1532.MethodsBreast cancer MDA-MB 231 and MCF-7 cells were cultured in DMEM (Dulbecco’s modified eagle’s media) with 0, 1 or 4.5 g/l of glucose. The telomerase activity was measured via quantitative Real-Time PCR, and the two telomerase subunits were semi-quantified by RT-PCR. Proliferation test and mitochondrial metabolism were assessed via tetrazolium salt reduction and cell counts; apoptosis was assessed via caspase-3 quantification and flow cytometry.ResultsA decrease in the telomerase activity of more than 75% was associated with a significant reduction in the mRNA expression of its catalytic subunit hTERT (Reverse Transcriptase) and a decrease in the mitochondrial metabolism by more than 80% under restricted glucose conditions. In addition, GR increased the effect of BIBR 1532. Glucose deprivation induces apoptosis via BIBR 1532-mediated telomerase inhibition in triple negative breast cancer cells, as assessed by caspase-3 measurements and Annexin analysis.ConclusionsTaken together, our results suggest that the effect of BIBR 1532 is potentiated by GR to induce triple negative breast cancer cell death.

Cellular response to rare earth mixtures (La and Gd) as components of degradable Mg alloys for medical applications

Rare earth (RE) elements have been proposed to improve the corrosion resistance of degradable Mg alloys for medical applications. However, good biocompatibility of the elements released by Mg alloys during degradation is essential for their use in implants. Most studies are focused on material science and engineering aspects, but the effects of ions released at the biological interface are not frequently addressed. The aim of this study was to contribute to the knowledge of in vitro toxicological effects of two RE Mg-alloying elements, La and Gd, as individual ions and in mixtures with and without Mg ions. Different combinations (Mg+Gd, Mg+La, and Mg+Gd+La) were used to evaluate their possible synergistic effects on CHO-K1 cells. Two sets of experiments were designed to assess (1) the cyto-genotoxic effect of La and Gd ions by neutral red (NR) technique, Reduction of tetrazolium salt (MTT), Viability with Acridine Orange staining, Clonogenic test, and Comet assay; and, (2) the possible synergistic toxicological effect of La and Gd ions in mixtures, and the influence of osmolarity increase on cellular response. Cytotoxic effects of RE were found at concentrations ≥200μM RE while DNA damage was detected for doses ≥1500μM and ≥1600μM for La and Gd, respectively. When mixtures of ions were evaluated, neither synergistic cytotoxic effects nor biological damage related to osmolarity increase were detected.

Toxicity assessment of organomodified clays used in food contact materials on human target cell lines

Nowadays, the incorporation of organomodified clays based on montmorillonite into polymers intended for packaging industry is a reality. The final result is a polymer nanocomposite with enhanced barrier properties. Different organomodified clays are already commercially available and others new ones are being developed; however little is known about their safety.In the present work, the cytotoxic effects (a tetrazolium salt reduction and protein content) of three organomodified clays, Cloisite®20A, a commercial clay, and Clay 1 and Clay 2, two novel modified clays developed by the Packaging, Transport, & Logistics Research Institute, were evaluated in Caco-2 and HepG2 cells after 24 and 48h of exposure. Our results showed that only Clay 2 induced toxic effects in both cell lines. The mean effective concentration was calculated for each case, showing Caco-2 to be more sensitive than HepG2. Moreover, in order to elucidate the toxicity mechanisms of Clay 2, different mechanistic biomarkers were investigated. Interleukin leakage and generation of intracellular reactive oxygen species were not observed, whereas glutathione content decreased in HepG2. DNA damage (comet assay) was induced in both cell lines at the highest concentration tested. Overall, results show that the type of clay, the concentrations range and the type of cell line play an important role in the toxicity observed.

Short- and long-term effects of silver nanoparticles on human microvascular endothelial cells.

To study the response to silver nanoparticles (Ag NP) of human microvascular endothelial cells, protagonists of angiogenesis. We cultured human microvascular endothelial cells and endothelial colony-forming cells in their corresponding growth medium. Stock solutions of Ag NP were prepared in culture medium and sonicated before use. They were added at different concentrations and for different times to culture media. The toxicity of Ag NP was investigated by measuring the reduction of yellow tetrazolium salt to dark purple formazan (MTT assay) at 575 nm. After staining with trypan blue, cell proliferation was assessed by counting viable cells. The lactate dehydrogenase leakage assay was performed on culture media by following the oxidation of NADH to NAD+ and monitoring the reaction kinetically at 340 nm. Reactive oxygen species production was quantified using 2'-7'-dichlorofluorescein diacetate. The alkaline comet assay was performed after mixing the cells with low melting-point agarose. Electrophoresis was then conducted and the samples were stained with ethidium bromide and analyzed with a fluorescence microscope. Ag NP are cytotoxic in a dose and time dependent fashion for HMEC. At high concentrations, Ag NP determine loss of membrane integrity as demonstrated by the increased activity of lactate dehydrogenase in the culture medium. Ag NP rapidly stimulate the formation of free radicals. However, pre-incubation with Trolox, apocynin, or N-acetyl-L-cysteine, antioxidants which have different structure and act through different mechanisms, is not sufficient to prevent cytotoxicity. Ag NP also induce DNA damage dose-dependently, as shown by comet assay. When exposed to sublethal concentrations of Ag NP for long times, the cells remain viable but are growth retarded. Interestingly, removal of Ag NP partially rescues cell growth. Also genotoxicity is reversible upon removal of Ag NP from culture medium, suggesting that no permanent modifications occur. It is noteworthy that Ag NP are cytotoxic and genotoxic also for endothelial progenitors, in particular for endothelial colony-forming cells, which participate to angiogenesis. Silver nanoparticles are cytotoxic and genotoxic for human microvascular endothelial cells and might become a useful tool to control excessive angiogenesis.

Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates

Echinocandin-resistant clinical isolates of Candida albicans have been reported, and key-hot spot mutations in the FKS1 gene, which encodes a major glucan synthase subunit, have been identified in these (caspofungin-resistant [CAS-R]) strains. Although these mutations result in phenotypic resistance to echinocandins in planktonic cells, there is little data on antifungal susceptibilities of CAS-R C. albicans strains within biofilms. Thus, we analyzed biofilms formed by 12 C. albicans CAS-R clinical strains in which we previously identified FKS1 hot-spot mutations and compared the sessile antifungal and paradoxical activity of anidulafungin (ANID), caspofungin (CAS), and micafungin (MICA). Biofilms were formed in a 96-well static microplate model and assayed using both tetrazolium-salt reduction and crystal violet assays, as well as examination by scanning electron microscopy. We first sought to assess biofilm formation and structure in these fks1 mutants and found that the biofilm mass and metabolic activities were reduced in most of the fks1 mutants as compared with reference strain SC5314. Structural analyses revealed that the fks1 mutant biofilms were generally less dense and had a clear predominance of yeast and pseudohyphae, with unusual "pit"-like cell surface structures. We also noted that sessile minimum inhibitory concentrations (MICs) to ANID, CAS, and MICA were higher than planktonic MICs of all but one strain. The majority of strains demonstrated a paradoxical effect (PE) to particular echinocandins, in either planktonic or sessile forms. Overall, biofilms formed by echinocandin-resistant clinical isolates demonstrated varied PEs to echinocandins and were structurally characterized by a preponderance of yeast, pseudohyphae, and pit-like structures.

Effect of antimicrobial agents incorporated into resilient denture relines on the Candida albicans biofilm

The antimicrobial action of five drugs incorporated in temporary denture relines on the fungal biofilm was evaluated. A Candida albicans biofilm (SC5314) was formed on specimens (10 × 1 mm) of materials (Trusoft and Softone) modified or not (control) by the drugs (nystatin, miconazole, ketoconazole, itraconazole, and chlorhexidine diacetate). Cell viability was determined spectrophotometrically by the tetrazolium salt reduction assay (XTT) after 24 h, 48 h, and 7 and 14 days of incubation. The minimum inhibitory concentrations (MICs) were those which inhibited 90% or more of fungal growth. Fungal susceptibility was confirmed by confocal laser scanning microscopy analysis. The MICs of drugs incorporated in the materials were 0.032, 0.256, 0.128, 0.256, and 0.064 g ml(-1) for nystatin, miconazole, ketoconazole, itraconazole, and chlorhexidine, respectively. Images from nystatin, chlorhexidine, and ketoconazole demonstrated no viable cells. The antimicrobials incorporated in the resilient materials inhibited fungal growth during 14 days, with lower MICs for nystatin and chlorhexidine.

Cytotoxicity and morphological effects induced by carvacrol and thymol on the human cell line Caco-2

Essential oils used as additives in the food industry due to its flavour, antimicrobial and antioxidant properties. Therefore, human can be exposed orally to these compounds through the ingestion of foods. In this sense, the present work aims to assess toxicological effects of oregano essential oil on the digestive tract. In concrete, the cytotoxic effects of two components of the oregano essential oils, carvacrol and thymol, and their mixture, on the intestinal cells line Caco-2 after 24 and 48h of exposure are studied. The basal cytotoxicity endpoints assayed (total protein content, neutral red uptake and the tetrazolium salt reduction) and the annexin/propidium iodide staining indicated that carvacrol and the mixture carvacrol/thymol induced toxic effects. Moreover, a morphological study was performed in order to determine the ultrastructural cellular damages caused by these substances. The main morphological alterations were vacuolated cytoplasm, altered organelles and finally cell death. In addition, although no cytotoxic effects were recorded for thymol at any concentration and time of exposure, ultrastructural changes evidenced cellular damage such as lipid degeneration, mitochondrial damage, nucleolar segregation and apoptosis.

Toxic effects of a modified montmorillonite clay on the human intestinal cell line Caco-2

The incorporation of the natural mineral clay montmorillonite into polymeric systems enhances their barrier properties as well as their thermal and mechanical resistance, making them suitable for a wide range of industrial applications, e.g., in the food industry. Considering humans could easily be exposed to these clays due to migration into food, toxicological and health effects of clay exposure should be studied. In the present work, the cytotoxic effects induced by two different clays (the unmodified clay Cloisite(®) Na(+) , and the organically modified Cloisite(®) 30B) on Caco-2 cells were studied after 24 and 48 h of exposure. The basal cytotoxicity endpoints assessed were total protein content, neutral red uptake and a tetrazolium salt reduction. Our results showed that only Cloisite(®) 30B induced toxic effects. Therefore, the effects of subcytotoxic concentrations of this clay on the generation of intracellular reactive oxygen species, glutathione content and DNA damage (comet assay) were investigated. Results indicate that oxidative stress may be implicated in the toxicity induced by Closite(®) 30B, in regards of the increases in intracellular reactive oxygen species production and glutathione content at the highest concentration assayed, while no damage was observed in DNA. The most remarkable morphological alterations observed were dilated cisternae edge in the Golgi apparatus and nucleolar segregation, suggesting impairment in the secretory functions, which could be related to inhibition in the synthesis of proteins.

Intracellular distribution of antitumor Ru (II) compounds: The lysosome and the lysosomal enzymes as targets for anticancer metal-based drugs

The therapeutic usefulness of anticancer agents relies on their ability to kill cancer cells while sparing normal cells and tissues. Ruthenium complexes hold great potential as suitable alternative drugs to the current cisplatin in the treatment of cancer. Our approach in this field has been the study of organoruthenium complexes, [RuII(η5-Cp)] family, namely [RuII(η5-Cp)(bipy)(PPh3)]+ (PPh3 = triphenylphosphine; bipy = 2,2’-bipyridine), TM34. This compound was tested for antitumor activity against tumor cells representative of human cancer diseases. A large spectrum of activity was found, largely surpassing cisplatin in efficacy (Fig 1).How a drug distributes and localizes within cells is of fundamental importance as the drug must concentrate in the compartment that houses its target(s). The importance of cellular enzymes on the effects of anticancer drugs for which multiple biological pathways have been proposed led us to address the involvement of the lysosomes and lysosomal enzymes (AcPases) in the mechanism of cell death.The cytotoxicity of TM34 was tested using the MTT assay. TM34 cellular distribution was measured by ICP-MS. The effect of TM34 on the activity of acid phosphatase (AcPase) was evaluated by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol. AcPase localization was investigated by TEM using the Record and Griffing (1988) cerium-based method.Results showed that the sensitivity of AcPase assay is higher in comparison to cell proliferation assays based on the reduction of tetrazolium salts (MTT) (Fig 2). From TEM images AcPase reaction product appears as dark electron dense deposits in the lysosomes and dictiosomes. After 3h treatment TM34 provokes disruption and vesiculation of the Golgi apparatus, although some reaction product is present in the lysosomes (Fig 2).The lysosome could be considered a possible target for TM34. Morphological evidence was observed for the participation of the Golgi apparatus in the vesiculation induced by the compound.Supported by the Fundação para a Ciência e Tecnologia (FCT): PTDC/QUI-QUI/101187/2008.