Comparison of in vitro cell cytotoxic assays for tumor necrosis factor

Vital dye uptake and postfixation dye assays have recently been used to examine the interaction between short-term (24-48 h) exposures to the monokine, tumor necrosis factor (TNF), and hyperthermic treatments with the finding that synergistic increases in cytotoxicity occurred. However, survival measured by these short-term dye assays is not necessarily closely related to eventual loss of clonogenic capacity. Treatment-induced growth delays, delayed cytotoxic effects, or perturbations of vital dye sequestration mechanisms could result in a different measurement of surviving fraction than given by a clonogenic assay. In this study we directly compared the neutral red vital dye uptake and clonogenic survival assays and confirmed in both assays that TNF-sensitive (L-929) and TNF-resistant (EMT-6) phenotypes show greatly reduced survival when treated with combined recombinant human TNF (1.0-0.0005 micrograms/ml) and hyperthermia (1-2 h at 43 degrees C). Moreover, we confirmed that sensitization of the TNF-resistant EMT-6 cells was largely dependent on monokine treatment before hyperthermia and was reduced by the reverse sequence. The greatest sensitization of TNF-responsive L-929 cells also occurred when TNF treatment preceded heating. These results for clonogenic survival are consistent with the hypothesis that hyperthermia used in combination with TNF in vivo is more cytotoxic than TNF or hyperthermia separately.

Effect of high polyol concentrations on the neutral red absorption assay and tetrazolium-MTT test of rat hepatocytes in primary culture

The Yatapoxviruses encode a distinct class of secreted TNF-binding protein (TNF-BP) that resembles an MHC class I heavy chain but distinct from any other known TNF inhibitor. Characterization of these viral TNF inhibitors from Tanapox virus, Yaba monkey tumor virus (YMTV) and a closely related version from Swinepox virus revealed dramatically differential TNF binding specificities for different mammalian species. The Tanapox virus 2L protein (TPV-2L) formed inhibitory complexes with human TNF, and interacted with monkey and canine TNF with high affinity but rabbit TNF with low affinity. On the other hand, YMTV-2L bound human and monkey TNF with high affinity but rabbit TNF with only low affinity. The TNF-BP from swinepox virus (SPV003/148) only interacted with porcine TNF with high affinity. The observed TNF binding analysis mirrored the biological activity of these TNF-binding protein to block TNF-induced cellular cytolysis. TPV-2L and YMTV-2L also inhibited the human TNF-mediated signaling in cells but TPV-2L exhibited higher affinity for human TNF (KD, 43 pm) compared with monkey (KD, 120 pm) whereas for YMTV-2L, the affinities were reversed (human TNF KD, 440 pm; monkey TNF KD, 230 pm). The interaction domain of human TNF with TNF-binding proteins is significantly different from that of TNFRs, as determined using human TNF mutants. We conclude that these poxvirus TNF-binding proteins represent a new class of TNF inhibitors and are distinct from the viral TNF receptor homologues characterized to date.

Microtiter plate radioreceptor assay for tumor necrosis factor and its receptors in large numbers of samples

Effect of anticoagulants and heat on the detection of tumor necrosis factor in murine blood

Studying the cyto-genotoxic effects of 12 cigarette smoke condensates on human lymphoblastoid cell line in vitro

MURINE IL-2 SECRETING RECOMBINANT BACILLUS CALMETTE-GUÉRIN AUGMENTS MACROPHAGE-MEDIATED CYTOTOXICITY AGAINST MURINE BLADDER CANCER MBT-2

Using a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extracellular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monoclonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.

Colleagues?Much has been published about production of tumor necrosis factor (TNF) by human macrophages. It is thought that blood sampling for TNF must be done with great care using a pyrogene-free anticoagulant and blood-collecting system. Then plasma or serum must be rapidly removed from cells and cooled before assay for TNF [1]. TNF has been implicated in the pathogenesis of several infectious and inflammatory processes including cerebral malaria [2]. Because large amounts of chloroquine circulate in the population living in areas endemic for malaria and the fact that this drug easily penetrates macrophages, we investigated the influence of chloroquine on TNF production by human macrophages in vitro. Blood was collected from healthy human nonimmune individuals, and monocytes were extracted on a density gradient followed by adherence to plastic dishes, as previously described [3]. Monocytes/macrophages were preincubated for 2 h with various amounts of chloroquine (diphosphate salt; Sigma Chemicals, St. Louis) corresponding to those observed in vivo. Cells were then stimulated with lipopolysaccharide (Escherichia coli 055:B5; Calbiochem-Behring, La Jolla, CA). After overnight incubation, TNF levels were measured in the supernatant by immunoradiometric assay (IRMA; Medgenix, Fleurus, Belgium). With chloroquine, we observed a dose-dependent significant decrease in TNF production (figure 1). The mechanism remains unknown.

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.

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.

To evaluate the cytotoxicity of mercury in dental amalgams, a stress protein assay was performed and the results were compared with the cytotoxicity evaluated by a neutral red uptake assay. The induction of a major stress protein, hsp70, was analyzed at levels of mRNA, synthesis and accumulation in human HeLa cells treated with extracts from amalgam, metal mercury and mercuric chloride. Mercuric chloride induced an increase in the synthesis of hsp70 at concentrations of mercury half those used for the neutral red uptake assay. The extracts from dental amalgam and metal mercury induced an increase in hsp70 mRNA at concentrations of mercury half those causing the inhibition of neutral red uptake into cells. Furthermore, the extracts from dental amalgam or metal mercury increased the synthesis of hsp70 and inhibited the uptake of dye at concentrations of mercury 1/10-1/50 lower than those at which mercuric chloride acted. These results suggest that the stress protein assay is more sensitive than the conventional neutral red assay for the evaluation of the cytotoxicity of mercury in dental amalgams and that the methods used in the preparation of metal solutions seem to be critical to the evaluation of cytotoxicity of dental materials.

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells.Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734-741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.

Journal Article Chloroquine Inhibits Tumor Necrosis Factor Production by Human Macrophages In Vitro Get access Stéphane Picot, Stéphane Picot Search for other works by this author on: Oxford Academic PubMed Google Scholar François Peyron, François Peyron Search for other works by this author on: Oxford Academic PubMed Google Scholar Jean-Philippe Vuillez, Jean-Philippe Vuillez Search for other works by this author on: Oxford Academic PubMed Google Scholar Benoît Polack, Benoît Polack Search for other works by this author on: Oxford Academic PubMed Google Scholar Pierre Ambroise-Thomas Pierre Ambroise-Thomas Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 164, Issue 4, October 1991, Page 830, https://doi.org/10.1093/infdis/164.4.830 Published: 01 October 1991

Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC-1 ( Poeciliopsis lucida)