Online monitoring of cell metabolism to assess the toxicity of nanoparticles: the case of cobalt ferrite

Different in vitro assays are successfully used to determine the relative cytotoxicity of a broad range of compounds. Nevertheless, different research groups have pointed out the difficulty in using the same tests to assess the toxicity of nanoparticles (NPs). In this study, we evaluated the possible use of a microphysiometer, Bionas 2500 analyzing system Bionas GmbH?, to detect in real time changes in cell metabolisms linked to NPs exposure. We focused our work on response changes of fibroblast cultures linked to exposure by cobalt ferrite NPs and compared the results to conventional in vitro assays. The measurements with the microphysiometer showed a cobalt ferrite cytotoxic effect, confirmed by the Colony Forming Efficiency assay. In conclusion, this work demonstrated that the measurement of metabolic parameters with a microphysiometer is a promising method to assess the toxicity of NPs and offers the advantage to follow on-line the cell metabolic changes.     

The Bionas technology for anticancer drug screening

Background: The Bionas 2500^® analyzing system is an advanced label-free technology using a cell-based multi-sensor array, which is commercially available. Data on metabolism, respiration, adhesion, cell proliferation and cell death rates, as well as ligand–receptor interactions (multi-parametric) can be acquired and statistically evaluated. Noteworthy is the possibility of analyzing later after effects and/or recovery after drug treatment. In addition, Bionas supports all conceivable drug application modes (one, two or more drugs). Specimens for drug screening with Bionas consist of human permanent cell lines, primary cell cultures as well as ‘tumor slices’ obtained from biopsies and surgery material. Objective/methods: Examples of measurements are presented and discussed. Conclusion: Although drug throughput is modest, the high quality of the information allows in-depth evaluation.     

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR.The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.     

Preservation,induction or incorporation of metabolism into the in vitro cellular system − Views to current opportunities and limitations

Metabolism plays a major role in the toxicokinetics of a vast majority of substances, although other dispositional processes have to be considered as well. There are currently a large repertoire of primary or permanent cells/cell lines with variable metabolic capacities and a number of experimental approaches to preserve, induce or incorporate biotransformation enzymes for the development of metabolically competent cells. Many of these cell lines possess also other important dispositional characteristics mimicking the in vivo situation. Such cell models can be employed in studies targeted for estimating metabolic disposition of a substance or the production of active metabolites and ensuing toxic end points. There are also ways to collect metabolic information by using a large number of non-cellular systems and build a coherent view on metabolism, although not really replacing the actual cellular system. Early consideration of metabolic competence is a necessary prerequisite for the validation and use of cellular systems for toxicity studies and in vitro–in vivo extrapolation.     

Examination of leg volume of patients for determining the therapeutic effect of 1-phenylephrine hydrochloride (author's transl)]

Distorsions and other injuries of a more or less grave kind are often found in combination with exudative processes. The anti-exudative effect of adequate drugs has so far been tested only on the rat paw oedema. As results gained from animal tests cannot immediately be applied to man, tests with human subjects were carried out. With a device by Diebschlag it has become possible to fulfil the demands of a simple but still exact measuring process for quantification of leg volumes. 12 subjects with a distorsion of the ankle joint were treated with a gel (active substance: 1-phenylephrine HCl) which reduces the volue of the leg and with a similar placebo gel, and the effects of both treatments were compared and examined. The analysis of data yielded statistically highly significant values for the effectivity of the active substance gel.     

Growth inhibition and induction of G(1) phase cell cycle arrest in neuroblastoma SH-SY5Y cell by tri-ortho-cresyl phosphate

It has been known that tri-ortho-cresyl phosphate (TOCP) can induce delayed neurotoxicity in humans and sensitive animal species; however, it also has influence on the developing central nervous system or differentiating neuronal cells. In this study, the effects of TOCP on cell proliferation and cell cycle regulation and the mechanisms that contribute to this effect were investigated by using human neuroblastoma SH-SY5Y cell line. Treatment of the cells with TOCP suppressed cell proliferation and reduced cell viability in a dose- and time-dependent manner. Analysis of cell cycle profile indicated that TOCP blocked cell cycle progression by arresting the cell cycle at G(1) phase. The data of determination of cell cycle regulated molecules at mRNA and protein levels showed that TOCP decreased cyclin D1 and increased p21 expression, while did not affect the p53 and p27 levels. Thus, these results indicated that TOCP might induce potential neurodevelopmental toxicity, and a possible mechanism of this toxicity might be the disturbance of cell proliferation by disrupting cell cycle regulatory proteins cyclin D1 and p21 expression.     

Liver function assessment by drug metabolism

Liver function can be assessed by administering an exogenous substance to quantify changes in hepatic blood flow, uptake, biotransformation, and excretion. Characterization of drug half-life, clearance, and product formation rates are possible methods for measuring hepatic efficiency. Allopurinol and caffeine have been used to measure metabolite formation followed by renal elimination of both parent substance and metabolite. Sorbitol, a substance with high intrinsic clearance, can reflect liver blood flow, while trimethadione, a low-extraction drug, has been used to measure liver enzyme capacity. Metabolites from lidocaine, methacetin, and aminopyrine have been measured in serum, urine, and breath tests. Salivary clearance measurements of caffeine and antipyrine are reported as suitable for routine use. Genetic diversity of isoenzymes and the many metabolic processes used by hepatocytes make it extremely difficult to quantify functional changes with one substance. Combinations of model substrates have been suggested to assess the many hepatic processes.     

Effects of flavonoids on immune and inflammatory cell functions.

No doubt can remain that the flavonoids have profound effects on the function of immune and inflammatory cells as determined by a large number and variety of in vitro and some in vivo observations. That these ubiquitous dietary chemicals may have significant in vivo effects on homeostasis within the immune system and on the behavior of secondary cell systems comprising the inflammatory response seems highly likely but more work is required to strengthen this hypothesis. Ample evidence indicates that selected flavonoids, depending on structure, can affect (usually inhibit) secretory processes, mitogenesis, and cell-cell interactions including possible effects on adhesion molecule expression and function. The possible action of flavonoids on the function of cytoskeletal elements is suggested by their effects on secretory processes. Moreover, evidence indicates that certain flavonoids may affect gene expression and the elaboration and effects of cytokines and cytokine receptors. How all of these effects are mediated is not yet clear but one important mechanism may be the capacity of flavonoids to stimulate or inhibit protein phosphorylation and thereby regulate cell function. Perhaps the counterbalancing effect of cellular protein tyrosine phosphatases will also be found to be affected by flavonoids. Some flavonoid effects can certainly be attributed to their recognized antioxidant and radical scavenging properties. A potential mechanism of action that requires scrutiny, particularly in relation to enzyme inhibition, is the redox activity of appropriately configured flavonoids. Finally, in a number of cell systems it seems that resting cells are not affected significantly by flavonoids but once a cell becomes activated by a physiological stimulus a flavonoid-sensitive substance is generated and interaction of flavonoids with that substance dramatically alters the outcome of the activation process.     

Modulatory roles of glycolytic enzymes in cell death

Cancer cells depend on an altered energy metabolism characterized by increased rates of both glycolysis and glutaminolysis. Accordingly, corresponding key metabolic enzymes are overexpressed or hyperactivated. As a result, this newly acquired metabolic profile determines most other cancer hallmarks including resistance to cell death. Recent findings highlighted metabolic enzymes as direct modulators of cell death pathways. Conversely, key mediators of cell death mechanisms are emerging as new binding partners of glycolytic actors; moreover, there is evidence that metabolic regulators re-localize to specific subcellular compartments or organelles to modulate various types of cell demise. The final outcome is the resistance against cell death programs. Current findings give a new meaning to metabolic pathways and allow understanding how they affect cancer-specific pathological alterations. Furthermore, they shed light on potentially targetable functions of metabolic actors to restore susceptibility of cancer cells to death. Here, we discuss an emerging interplay between cell metabolism and cell death, focusing on interactions that may offer new options of targeted therapies in cancer treatment involving more specifically hexokinases and glyceraldehyde-3-phosphate dehydrogenase.     

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20 nm and 200 nm) and titanium dioxide (21 nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact.     

Comprehensive two-dimensional APTES-decorated MCF7-cell membrane chromatographic system for characterizing potential anti-breast-cancer components from Yuanhu–Baizhi herbal medicine pair

Rhizoma corydalis and Radix Angelicae Dahurica (Yuanhu–Baizhi) herbal medicine pair has been used for thousands of years and has been reported to be potentially active in recent cancer therapy. But the exact active components or fractions remain unclear. In this study, a new comprehensive two-dimensional (2D) 3-aminopropyltriethoxysilane (APTES)-decorated MCF7-cell membrane chromatography (CMC)/capcell-C18 column/time-of-flight mass spectrometry system was established for screening potential active components and clarifying the active fraction of Yuanhu–Baizhi pair. APTES was modified on the surface of silica, which can provide an amino group to covalently link cell membrane fragments with the help of glutaraldehyde in order to improve the stability and column life span of the MCF7 CMC column. The comprehensive 2D MCF7-CMC system showed good separation and identification abilities. Our screen results showed that the retention components are mainly from the alkaloids in Yuanhu (12 compounds) and the coumarins (10 compounds) in Baizhi, revealing the active fractions of Yuanhu–Baizhi herbal medicine pair. Oxoglaucine, protopine, berberine, osthole, isopimpinellin and palmitic acid were selected as typical components to test the effects on cell proliferation and their IC₅₀ were calculated as 38.17 μM, 29.45 μM, 45.42 μM, 132.7 μM, 156.8 μM and 90.5 μM respectively. Cell apoptosis assay showed that the drug efficacy was obtained mainly through inducing cell apoptosis. Furthermore, a synergistic assay results demonstrated that oxoglaucine (representative of alkaloids from Yuanhu) and isopimpinellin (representative of coumarins from Baizhi) showed significant synergistic efficacy with GFT, indicating that these components may act on other membrane receptors. The proposed 2D CMC system could also be equipped with other cells for further applications. Besides, the follow-up in-vitro experimental strategy using cell proliferation assay, cell apoptosis assay and synergistic assay proved to be a practical way to confirm the active fractions of herbal medicine.     

Prediction of LD50 values by cell culture

Alternative approaches to toxicity tests with animals-more precisely defined as complementary methods-serve for reducing the number of animal experiments. The determination of cytotoxicity with cell cultures could be an important complementary method to get more information about a general basal toxicity of a substance in an organism during the course of an acute toxicity experiment. We employed the endothelial cell line BKEz-7 in a general (i.e. nonspecific) cytotoxicity test. As a quantitative marker of cytotoxic effects we determined the inhibition concentration for a fifty per cent reduction of the cell number per culture (IC50). the IC50 values of 28 compounds were compared with the corresponding LD50 values in the acute toxicity experiments. In these comparisons we included the determination of the lethality index (LI = IC50/LD50), the correlation analyses and the application of a single linear regression model. The following results were obtained. Between the IC50 and LD50 values a positive correlation exists at the significance level 1 - alpha = 0.95. After plotting the regression line in a coordinate system, a novel graphic prediction of LD50 estimations is possible with a relatively high accuracy. On the basis of tenfold steps of concentrations a new scale of cytotoxicity was attributed to the internationally employed classification of LD50. In the cytotoxicity test with the cell line BKEz-7 a broad spectrum of substance classes can be tested. The cytotoxicity test and the comparison of IC50 and LD50 p.o. as described here contribute to reduce the number of animals in the acute toxicity experiments.     

Effects of haloperidol, clozapine and olanzapine on the survival of human neuronal and immune cells in vitro

Cytotoxic effects on neuronal as well as on immune cells have been reported for both typical and atypical antipsychotic drugs. We evaluated the effects of different concentrations of a typical (haloperidol) and two atypical (clozapine, olanzapine) antipsychotics on the survival of human neuronal (SH-SY5Y cells) and immune cells (U937 cells) by determining the metabolic activity after 24 h of incubation by the modified tetrazolium method. The dopaminergic neuroblastoma SH-SY5Y and the lymphoma U-937 cell line are well established models for in vitro investigations. To further elucidate possible mechanisms of action we also determined the ATP content in the cultured cells. After experimental treatment, significant effects were detected by Kruskal Wallis test for all treatment conditions. Post-hoc tests (Dunn's method) showed that haloperidol and clozapine at the two highest concentrations (25 and 50 microg/ml) caused a significant decrease of metabolic activity in both cell systems, which was also detectable after treatment with clozapine at a concentration of 12.5 microg/ml in U937 cells. In contrast, olanzapine induced a significant increase in metabolic activity of SH-SY5Y cells at all concentrations except for the concentration of 3.1 microg/ml, whereas the metabolic activity in U937 cells was increased at concentrations of 1.6 and 6.25 microg/ml. For the determination of ATP content, the LD(50) values of the metabolic activity were used, except for olanzapine for which no distinct LD(50) value was available. Significant changes were detected for all treatments and post-hoc tests revealed that haloperidol caused a significant decrease compared to the control condition in both cell systems. These findings suggest that antipsychotic substances of different classes exert differential metabolic effects in both neuronal and immune cell systems.     

Quantitative Glucose and ATP Sensing in Mammalian Cells

The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic dysfunction plays an important role in many human diseases, including diabetes, cancer, inherited mitochondrial disorders, and metabolic syndrome. The monosaccharide glucose constitutes a key source of cellular energy. Following its import across the plasma membrane, glucose is converted into pyruvate by the glycolysis pathway. Pyruvate oxidation supplies substrates for the ATP-generating mitochondrial oxidative phosphorylation (OXPHOS) system. To gain cell-biochemical knowledge about the operation and regulation of the cellular energy metabolism in the healthy and diseased state, quantitative knowledge is required about (changes in) metabolite concentrations under (non) steady-state conditions. This information can, for instance, be used to construct more realistic in silico models of cell metabolism, which facilitates understanding the consequences of metabolic dysfunction as well as on- and off-target effects of mitochondrial drugs. Here we review the current state-of-the-art live-cell quantification of two key cellular metabolites, glucose and ATP, using protein-based sensors. The latter apply the principle of FRET (fluorescence resonance energy transfer) and allow measurements in different cell compartments by fluorescence microscopy. We further summarize the properties and applications of the FRET-based sensors, their calibration, pitfalls, and future perspectives.     

Effects of ginsenosides Rg<Subscript>3</Subscript> and Rh<Subscript>2</Subscript> on the proliferation of prostate cancer cells

Ginseng has an anti-cancer effect in several cancer models. This study was to characterize active constituents of ginseng and their effects on proliferation of prostate cancer cell lines, LNCaP and PC3. Cell proliferation was measured by [3H]thymidine incorporation, the intracellular calcium concentration by a dual-wavelength spectrophotometer system, effects on mitogen-activated protein (MAP) kinases by Western blotting, and cell attachment and morphologic changes were observed under a microscope. Among 11 ginsenosides tested, ginsenosides Rg3 and Rh2 inhibited the proliferation of prostate cancer cells. EC50s of Rg3 and Rh2 on PC3 cells were 8.4 microM and 5.5 microM, respectively, and 14.1 microM and 4.4 microM on LNCaP cells, respectively. Both ginsenosides induced cell detachment and modulated three modules of MAP kinases activities differently in LNCaP and PC3 cells. These results suggest that ginsenosides Rg3 and Rh2-induced cell detachment and inhibition of the proliferation of prostate cancer cells may be associated with modulation of three modules of MAP kinases.     

A rapid screening method to test apoptotic synergisms of ochratoxin A with other nephrotoxic substances.

The body may be exposed simultaneously to more than one nephrotoxic substance and to measure the effects of the great number of possible combinations of nephrotoxins will rapidly become a great challenge when using the traditional methods. Therefore, we developed a rapid and cost-efficient method to screen the apoptotic potential of combinations of known cell- or nephrotoxic substances as ochratoxin A (OTA), cisplatin, cadmium, H(2)O(2), and amphotericin B on renal epithelial cell lines. The cells were seeded in 96-well plates and the apoptotic and necrotic potential of different combinations of nephrotoxins was determined. We found different results for the combinations used: depending on the concentrations of the various substances, antagonistic, additive, or potentiating effects on caspase-3 activity were found after co-exposure to OTA. We conclude that the co-exposure of renal cells to OTA with other substances can enhance or reduce the apoptotic potential of one substance alone depending on the substance, the concentration and on the cell line investigated. A "harmless" substance can thus convert to a potent cell toxic substance when combined with OTA or vice versa. The underlying mechanisms of the synergistic effects remain unknown.     

Toxicological screening models: drug-induced oxidative hemolysis

In an attempt to obtain a simple screening system for the assessment of toxic-hemolytic effects of chemical substances, a battery of hematological tests was used. Phenacetin served as reference substance. The drug caused reversible formation of methemoglobin and Heinz bodies and an increase in peripheral reticulocytes after 2 and 4 weeks of treatment. Furthermore, an increase in the mean corpuscular volume of red blood cells (RBC) and the volume of RBC ghosts in hypotonic solutions, and a decrease of the mean corpuscular fragility was observed. The latter changes are considered to be a consequence of regenerative RBC compensation rather than due to structural membrane alteration caused by the drug. The results suggest that only a combination of several hematological tests can provide comprehensive information about the hemolytic potential of chemical substances, and that for screening purposes small numbers of animals are often sufficient.     

Mast cells in allergic and inflammatory diseases

Mast cells are important in the development of allergic and anaphylactic reactions, but also in acquired and innate immunity. There is also increasing evidence that mast cells participate in inflammatory diseases, where they can be activated by non-allergic triggers, such as neuropeptides and cytokines, often having synergistic effects as in the case of substance P (SP) and IL-33. Secretion of vasoactive mediators, cytokines and proteinases contribute to the development of coronary artery disease (CAD), as well as to diet-induced obesity and the metabolic syndrome. Mast cells may be able to orchestrate such different biological processes through their ability to release pro-inflammatory mediators selectively without the degranulation typical of allergic reactions. Recent evidence suggests that mitochondrial uncoupling protein 2 (UCP2) and mitochondrial translocation regulate mast cell degranulation, but not selective mediator release. Better understanding of these two processes and how mast cells exert both immunostimulatory and immunosuppressive actions could lead to the development of inhibitors of release of specific mediators with novel therapeutic applications.     

Early metabolic changes in response to lung injury: extrapolation from animals to humans

The pulmonary capillary endothelium provides a nonthrombogenic, semipermeable barrier between pulmonary blood and tissues. In recent years, particular attention has been focused on the ability of these cells to metabolize a variety of circulating biologically active substances either by interiorizing the substance through specific membrane transport processes or by directly altering the substance by way of enzymatic activity at the plasma membrane. Serotonin, norepinephrine, and several prostaglandins are examples of biologically active substances that are removed from the circulation by the pulmonary endothelium by way of specific transmembrane transport processes. Concomitant with the increased interest in metabolic functions of the pulmonary endothelium, there has been a growing awareness of the central role of endothelial cell abnormalities in the pathogenesis of various lung injuries and disease states. During the past several years, considerable evidence has accumulated in support of the hypothesis that alterations in the metabolic functions of the lung provide a method of detecting lung injury in vivo, and tests of the metabolic functions of the lung have progressed from in vitro systems to animal models to humans. This paper reviews some of the evidence responsible for this progression and discusses some of the limitations inherent in the extrapolation of lung metabolism studies from animal models to humans. In this discussion, particular emphasis is placed on the pulmonary uptake and metabolism of serotonin, norepinephrine, and prostaglandins E and F by mammalian lungs.     

Delphinidin induces apoptosis and inhibits epithelial‐to‐mesenchymal transition via the ERK/p38 MAPK‐signaling pathway in human osteosarcoma cell lines

Delphinidin is major anthocyanidin that is extracted from many pigmented fruits and vegetables. This substance has anti‐oxidant, anti‐inflammatory, anti‐angiogenic, and anti‐cancer properties. In addition, delphinidin strongly suppresses the migration and invasion of various cancer cells during tumorigenesis. Although delphinidin has anti‐cancer effects, little is known about its functional roles in osteosarcoma (OS). For these reasons, we have demonstrated the effects of delphinidin on OS cell lines. The effects of delphinidin on cell viability and growth of OS cells were assessed using the MTT assay and colony formation assays. Hoechst staining indicated that the delphinidin‐treated OS cells were undergoing apoptosis. Flow cytometry, confocal microscopy, and a western blot analysis also indicated evidence of apoptosis. Inhibition of cell migration and invasion was found to be associated with epithelial‐to‐mesenchymal transition (EMT), observed by using a wound healing assay, an invasion assay, and a western blot analysis. Furthermore, delphinidin treatment resulted in a profound reduction of phosphorylated forms of ERK and p38. These findings demonstrate that delphinidin treatment suppressed EMT through the mitogen‐activated protein kinase (MAPK) signaling pathway in OS cell lines. Taken together, our results suggest that delphinidin strongly inhibits cell proliferation and induces apoptosis. Delphinidin treatment also suppresses cell migration and prevents EMT via the MAPK‐signaling pathway in OS cell lines. For these reasons, delphinidin has anti‐cancer effects and can suppress metastasis in OS cell lines, and it might be worth using as an OS therapeutic agent.