MTT Reduction Activity Research Articles

Patterns of MTT reduction in mammalian spermatozoa.

MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.

Phenyl Saligenin Phosphate Disrupts Cell Morphology and the Actin Cytoskeleton in Differentiating H9c2 Cardiomyoblasts and Human-Induced Pluripotent Stem-Cell-Derived Cardiomyocyte Progenitor Cells.

We have previously shown that phenyl saligenin phosphate (PSP), an organophosphorus compound which is classed as a weak inhibitor of acetylcholinesterase, triggered cytotoxicity in mitotic and differentiated H9c2 cardiomyoblasts. The aim of this study was to assess whether sublethal concentrations of PSP could disrupt the morphology of differentiating rat H9c2 cardiomyoblasts and human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells (hiPSC-CMs) and to assess the underlying cytoskeletal changes. PSP-induced changes in protein expression were monitored via Western blotting, immunocytochemistry, and proteomic analysis. PSP-mediated cytotoxicity was determined by measuring MTT reduction, LDH release, and caspase-3 activity. Sublethal exposure to PSP (3 μM) induced morphological changes in differentiating H9c2 cells (7, 9, and 13 days), reflected by reduced numbers of spindle-shaped cells. Moreover, this treatment (7 days) attenuated the expression of the cytoskeletal proteins cardiac troponin I, tropomyosin-1, and α-actin. Further proteomic analysis identified nine proteins (e.g., heat shock protein 90-β and calumenin) which were down-regulated by PSP exposure in H9c2 cells. To assess the cytotoxic effects of organophosphorus compounds in a human cell model, we determined their effects on human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells. Chlorpyrifos and diazinon-induced cytotoxicity (48 h) was evident only at concentrations >100 μM. By contrast, PSP exhibited cytotoxicity in hiPSC-CMs at a concentration of 25 μM following 48 h exposure. Finally, sublethal exposure to PSP (3 μM; 7 days) induced morphological changes and decreased the expression of cardiac troponin I, tropomyosin-1, and α-actin in hiPSC-CMs. In summary, our data suggest cardiomyocyte morphology is disrupted in both cell models by sublethal concentrations of PSP via modulation of cytoskeletal protein expression.

Effects of octreotide on autophagy markers and cell viability markers related to metabolic activity in rat pituitary tumor cells.

The aim of this work was to investigate possible direct effects of the somatostatin analog octreotide on autophagy markers and markers of cellular metabolic activity using in vitro cultured rat pituitary tumor cells (GH3 cell line). We measured two markers of the autophagic flux in cell lysates by Western blot and MTT reductive activity, total cellular ATP levels, pyruvate dehydrogenase (PDH) complex activity in cells lysates as markers of cell viability related to metabolic activity. Octreotide (100nM) treatment induced autophagy activation (increased LC3-I protein lipidation) and enhanced the autophagic flux (SQSTM1/p62 protein downregulation) in GH3 cells in different incubation media, in detail in Hank's balanced salt solution (HBSS) as well as in maintenance medium with serum. We did not observe any decrease of redox activity and energy production related to the induction of autophagy by octreotide. On the other hand, short-term treatments with octreotide in HBSS tended to enhance MTT reduction activity and to increase PDH complex enzymatic activity and ATP levels measured in GH3 cell lysates. We provided evidence that octreotide can affect autophagy in pituitary tumor cells. The observed effects of octreotide were not related to a decrease of cellular metabolic activity. Finally, the induction of autophagy was either short-lived or overshadowed by other factors in the long term and this limit does not help clarifying their real impact on the pharmacological activity of somatostatin analogs.

Zinc interference with Cd‐induced hormetic effect in differentiated Caco‐2 cells: Evidence for inhibition downstream ERK activation

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium represents an effective protective barrier against Cd toxicity, but it is also a target tissue that may accumulate and trap high levels of the ingested metal. Using human enterocytic-like Caco-2 cells, we have previously shown that Cd may induce a concentration and time-dependent increase in 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT)-reducing activity in differentiated cultures with correlation to ERK1/2 activation. The present study shows that (a) Zn prevents the Cd-induced hormesis effect on MTT reduction in a concentration-dependent manner, without inhibiting Cd-induced ERK1/2 activation; (b) Zn also induces similar hormetic stimulation of MTT-reducing activity but without ERK1/2 activation. The effect of both metals was sensitive to inhibitors of translation during protein synthesis. There is evidence for the involvement of reactive oxygen species (ROS) in Cd-induced ERK1/2 activation. In contrast, the Zn effect on the MTT-reducing activity would not be triggered by ROS but it would be sensitive to the redox state of the cell. Steps downstream ERK1/2 activation by Cd does not involve eIF4E which is rather downregulated by Cd. In conclusion, Cd and Zn both can modify translation processes during protein synthesis via different signaling cascades with crosstalk, and cross-inhibition may occur. This phenomenon is observed over a small range of metal concentrations and is characterized by a hormesis-like response. Considering that the hormetic effect on dehydrogenase activity could reflect an adaptive response to the metals whether cross-inhibition is beneficial is an open question.

4,4-Diisothiocyanatostilbene Disulfonic Acid Enhanced 15-Deoxy-Δ12,14-prostaglandin J2-Induced Neuronal Apoptosis.

4,4-Diisothiocyanatostilbene disulfonic acid (DIDS), an antagonist of anion channel including voltage-dependent anion channel (VDAC), acts as both neurotoxicant and neuroprotectant, resulting in the controversy. VDAC contributes to neuronal apoptosis and is a candidate target protein of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). Caspase-3 is activated during neuronal apoptosis caused by 15d-PGJ2. In the present study, we ascertained whether DIDS was neuroprotective or neurotoxic in the primary culture of rat cortical neurons. Neuronal cell viabilities were primarily evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) reduction assay. Plasma membrane integrity and apoptosis were detected by the staining of propidium iodide (PI) and Hoechst33342, respectively. Alternatively, apoptosis was also measured by caspase-3 assay kit. DIDS did not prevent neurons from undergoing the 15d-PGJ2-induced apoptosis. In contrast, DIDS caused neuronal cell death in a concentration-dependent manner by itself, confirming its neurotoxicity. The sublethal application of DIDS did not decrease MTT-reducing activity, increase caspase-3 activity, condense chromatin, allow PI to enter neuron and degenerate neuronal morphology significantly. Interestingly, DIDS enhanced the 15d-PGJ2-induced neuronal apoptosis markedly under the sublethal condition. To our knowledge, this is the first report of synergistic effects of DIDS on the neurotoxicity of 15d-PGJ2.

Upregulation of connexin43 by glucose deprivation in H9c2 cells via the extracellular signal‑regulated kinase/mitogen‑activated protein kinase signaling pathway.

Cardiac connexin43 (Cx43) serves an essential role in maintaining the functional integrity of the heart. The present study investigated the effect of glucose deprivation (GD) on Cx43 protein expression levels in H9c2 cells, and demonstrated that following 2 h GD, Cx43 protein expression levels in H9c2 cells increased by ~68%. In addition, GD activated the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway, which regulated the expression levels of cardiac Cx43. A MAPK inhibitor and U0126, an ERK inhibitor, abolished the effects of GD on Cx43 expression levels. Under GD, the protein expression levels of Beclin-1, p62 and LC3 were augmented, and were decreased in the presence of ERK inhibitor or siRNA-ERK. In addition, H9c2 cells exposed to GD exhibited marked increase in LDH release and decreased MTT reduction activity, all of which were not significantly reversed by U0126 treatment. Therefore, the ERK/MAPK signaling pathway may be involved in elevating cardiac Cx43 expression levels under GD in H9c2 cells.

Effects of metformin on cell growth and AMPK activity in pituitary adenoma cell cultures, focusing on the interaction with adenylyl cyclase activating signals.

For a few years we have been investigating AMP-activated protein kinase (AMPK) as a target for drug therapy of GH-secreting pituitary adenomas. Aim of this study was to investigate the direct effects of metformin, which causes AMPK activation in different cell types, on rat pituitary adenoma cell growth and on related cell signalling pathways. Our results suggest that metformin can exert a growth-inhibitory activity in rat pituitary tumor cells mediated by AMPK activation, although multiple mechanisms are most likely involved. Membrane proteins, including growth factor receptors, are valuable targets of AMPK. The inhibition of the mTOR-p70S6 kinase signalling pathway plays a role in the suppressive effect of metformin on pituitary tumor cell growth. Metformin did not affect the MTT reduction activity in energetic stress conditions. Finally, metformin was still able to induce AMPK activation and to inhibit cell growth in cells treated with forskolin and in transfected cells overexpressing GHRH-receptor and treated with GHRH. Hence, adenylyl cyclase over-activation does not account for the lack of response of some human pituitary tumors to AMPK-activating compounds invitro.

Prostaglandin E2 facilitates neurite outgrowth in a motor neuron-like cell line, NSC-34

Prostaglandin E2 (PGE2) exerts various biological effects by binding to E-prostanoid receptors (EP1-4). Although recent studies have shown that PGE2 induces cell differentiation in some neuronal cells such as mouse DRG neurons and sensory neuron-like ND7/23 cells, it is unclear whether PGE2 plays a role in differentiation of motor neurons. In the present study, we investigated the mechanism of PGE2-induced differentiation of motor neurons using NSC-34, a mouse motor neuron-like cell line. Exposure of undifferentiated NSC-34 cells to PGE2 and butaprost, an EP2-selective agonist, resulted in a reduction of MTT reduction activity without increase the number of propidium iodide-positive cells and in an increase in the number of neurite-bearing cells. Sulprostone, an EP1/3 agonist, also significantly lowered MTT reduction activity by 20%; however, no increase in the number of neurite-bearing cells was observed within the concentration range tested. PGE2-induced neurite outgrowth was attenuated significantly in the presence of PF-0441848, an EP2-selective antagonist. Treatment of these cells with dibutyryl-cAMP increased the number of neurite-bearing cells with no effect on cell proliferation. These results suggest that PGE2 promotes neurite outgrowth and suppresses cell proliferation by activating the EP2 subtype, and that the cAMP-signaling pathway is involved in PGE2-induced differentiation of NSC-34 cells.

Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells.

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT), that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A). The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm) in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction.

Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity.

The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588]

Phenyl Saligenin Phosphate Induced Caspase-3 and c-Jun N-Terminal Kinase Activation in Cardiomyocyte-Like Cells.

At present, little is known about the effect(s) of organophosphorous compounds (OPs) on cardiomyocytes. In this study, we have investigated the effects of phenyl saligenin phosphate (PSP), two organophosphorothioate insecticides (diazinon and chlorpyrifos), and their acutely toxic metabolites (diazoxon and chlorpyrifos oxon) on mitotic and differentiated H9c2 cardiomyoblasts. OP-induced cytotoxicity was assessed by monitoring MTT reduction, LDH release, and caspase-3 activity. Cytotoxicity was not observed with diazinon, diazoxon, or chlorpyrifos oxon (48 h exposure; 200 μM). Chlorpyrifos-induced cytotoxicity was only evident at concentrations >100 μM. In marked contrast, PSP displayed pronounced cytotoxicity toward mitotic and differentiated H9c2 cells. PSP triggered the activation of JNK1/2 but not ERK1/2, p38 MAPK, or PKB, suggesting a role for this pro-apoptotic protein kinase in PSP-induced cell death. The JNK1/2 inhibitor SP 600125 attenuated PSP-induced caspase-3 and JNK1/2 activation, confirming the role of JNK1/2 in PSP-induced cytotoxicity. Fluorescently labeled PSP (dansylated PSP) was used to identify novel PSP binding proteins. Dansylated PSP displayed cytotoxicity toward differentiated H9c2 cells. 2D-gel electrophoresis profiles of cells treated with dansylated PSP (25 μM) were used to identify proteins fluorescently labeled with dansylated PSP. Proteomic analysis identified tropomyosin, heat shock protein β-1, and nucleolar protein 58 as novel protein targets for PSP. In summary, PSP triggers cytotoxicity in differentiated H9c2 cardiomyoblasts via JNK1/2-mediated activation of caspase-3. Further studies are required to investigate whether the identified novel protein targets of PSP play a role in the cytotoxicity of this OP, which is usually associated with the development of OP-induced delayed neuropathy.

Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 μM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

Chitosan as an adjuvant-like substrate for dendritic cell culture to enhance antitumor effects

To induce monocyte differentiation into dendritic cells (DCs) is the essential protocol for the DC-mediated cancer immunotherapy. In this study, monocytes isolated from mouse bone marrow were cultured on chitosan substrate to evaluate the effect of the chitosan culture system on the induction and tumor protection of DCs. Compared to tissue culture polystyrene (TCPS), the chitosan culture system could enhance monocyte aggregation and detachment with increased MTT reduction activity and expression of DC marker CD11c and LPS co-receptor CD14. Moreover, compared to TCPS, chitosan could enhance lipopolysaccharides (LPS)-stimulated DCs to secrete higher amount of IL-12. More importantly, vaccination of tumor lysate-pulsed DCs harvested from chitosan could increase cytotoxic T-lymphocyte (CTL) activity and showed significantly enhanced anti-tumor effect than those from TCPS. Therefore, the current study demonstrated that a protocol to culture DCs on a less-adherent chitosan substrate followed by treatment with tumor lysate has the potential in future DC-based vaccine application.

Effects of ethanol and acetaldehyde in zebrafish brain structures: An in vitro approach on glutamate uptake and on toxicity-related parameters

Ethanol (EtOH) and its metabolite, acetaldehyde (ALD), induce deleterious effects on central nervous system (CNS). Here we investigate the in vitro toxicity of EtOH and ALD (concentrations of 0.25%, 0.5%, and 1%) in zebrafish brain structures [telencephalon (TE), opticum tectum (OT), and cerebellum (CE)] by measuring the functionality of glutamate transporters, MTT reduction, and extracellular LDH activity. Both molecules decreased the activity of the Na+-dependent glutamate transporters in all brain structures. The strongest glutamate uptake inhibition after EtOH exposure was 58% (TE-1%), and after ALD, 91% (CE-1%). The results of MTT assay and LDH released demonstrated that the actions of EtOH and its metabolite are concentration and structure-dependent, in which ALD was more toxic than EtOH. In summary, our findings demonstrate a differential toxicity in vitro of EtOH and ALD in zebrafish brain structures, which can involve changes on glutamatergic parameters. We suggest that this species may be an interesting model for assessing the toxicological actions of alcohol and its metabolite in CNS.

Improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay for measuring the viability of lactic acid bacteria

The aim of this study was to adapt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay (MCA) (Wang et al., 2010) for determination of lactic acid bacteria cell count. Our study is helpful in developing protocols for measuring the viability of other lactic acid bacteria. We determined the cell concentration range which gives linear regression of CFU (colony forming units) and formazan production. In our experiment three authentic lactic acid bacteria strains were investigated: Lactobacillus rhamnosus VT1, L. plantarum 2142, and L. sakei DSM 2001. As data show beside the strain also the growth medium has influence on the MTT reduction activity, this means that for each special case separate calibration curve has to be prepared. Based on MTT reduction we also developed an improved microtiter plate assay which proved to be reliable for a rapid cell count determination. So our methods are only applicable for estimate cell number with fixed parameters, but under given circumstances they are fast and sensitive methods. With the modified methods we can rapidly measure the dehydrogenase enzyme activity of the lactic acid bacteria cells. With the microplate assay we can measure many conditions and strains at the same time.

Polybrominated diphenyl ethers enhance the production of proinflammatory cytokines by the placenta

Polybrominated diphenyl ether(s) (PBDE) are ubiquitous environmental contaminants that bind and cross the placenta but their effects on pregnancy outcome are unclear. It is possible that environmental contaminants increase the risk of inflammation-mediated pregnancy complications such as preterm birth by promoting a proinflammatory environment at the maternal–fetal interface. We hypothesized that PBDE would reduce IL-10 production and enhance the production of proinflammatory cytokines associated with preterm labor/birth by placental explants. Second-trimester placental explants were cultured in either vehicle (control) or 2 μM PBDE mixture of congers 47, 99 and 100 for 72 h. Cultures were then stimulated with 106 CFU/ml heat-killed Escherichia coli for a final 24 h incubation and conditioned medium was harvested for quantification of cytokines and PGE2. COX-2 content and viability of the treated tissues were then quantified by tissue ELISA and MTT reduction activity, respectively. PBDE pre-treatment reduced E. coli-stimulated IL-10 production and significantly increased E. coli-stimulated IL-1β secretion. PBDE exposure also increased basal and bacteria-stimulated COX-2 expression. Basal, but not bacteria-stimulated PGE2, was also enhanced by PBDE exposure. No effect of PBDE on viability of the explants cultures was detected. In summary, pre-exposure of placental explants to congers 47, 99, and 100 enhanced the placental proinflammatory response to infection. This may increase the risk of infection-mediated preterm birth by lowering the threshold for bacteria to stimulate a proinflammatory response(s).

Biological Behavior of Chitosan-Fibroin-Hydroxyapatite Scaffolds with STRO+1A, MC3T3-E1 and SaOS2 Cells<sup></sup>

Hybrid composites with chitosan (CHI), silk fibroin (SF) and hydroxyapatite (HA) are biocompatible and attractive for bone engineering applications. The objective of this work was to evaluate in vitro cells behavior in contact with CHI-SF-HA scaffolds. The scaffolds were produced from a chitosan solution (2%wt) with SF (1%wt) and HA powders (1%wt) in acetic acid. This solution was molded, frozen and the scaffolds were freeze-dried, crosslinked, and then, submitted to in vitro tests with STRO+1A, MC3T3-E1 and SaOS2 cells under static conditions for 7, 14 and 21 days. The scaffolds were characterized through X-ray diffraction (XRD), infrared spectroscopy (FTIR) and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Cell viability and activity was assessed by MTT reduction and alkaline phosphate (ALP) activity detection. XRD patterns showed characteristic peaks at 8.8º, 20.3º and 24.6º (corresponding to SF) and peaks at 31.8º, 32.2º and 32.9º (corresponding to HA). The FTIR presented characteristic bands of the amide groups (SF) and of the phosphate and carbonate groups (HA). The EDS showed the presence of the C, O, N, P and Ca elements. SEM analyses showed the scaffold morphology, and indicated cell growth across the surface of the sample. The STRO+1A and MC3T3-E1 cells presented the best cell adhesion. MTT assay showed an increase of the cell number with time and the 21 days analyses showed the best proliferation for SaOS2 cells (p<0.01), STRO+1A cells (p<0.04) and MC3T3-E1 cells, respectively. The ALP activity was higher at 21 days for all cells types and the SaOS2 cells presented the best results in all analyses (p<0.01). Molecular studies are under progress to evaluate more deeply the biocompatibility of these scaffolds. Moreover, dynamic studies in bioreactor are under progress to improve cell colonization inside scaffolds.

In vitro toxicity of particulate matter (PM) collected at different sites in the Netherlands is associated with PM composition, size fraction and oxidative potential - the RAPTES project

BackgroundAmbient particulate matter (PM) exposure is associated with respiratory and cardiovascular morbidity and mortality. To what extent such effects are different for PM obtained from different sources or locations is still unclear. This study investigated the in vitro toxicity of ambient PM collected at different sites in the Netherlands in relation to PM composition and oxidative potential.MethodPM was sampled at eight sites: three traffic sites, an underground train station, as well as a harbor, farm, steelworks, and urban background location. Coarse (2.5-10 μm), fine (< 2.5 μm) and quasi ultrafine PM (qUF; < 0.18 μm) were sampled at each site. Murine macrophages (RAW 264.7 cells) were exposed to increasing concentrations of PM from these sites (6.25-12.5-25-50-100 μg/ml; corresponding to 3.68-58.8 μg/cm2). Following overnight incubation, MTT-reduction activity (a measure of metabolic activity) and the release of pro-inflammatory markers (Tumor Necrosis Factor-alpha, TNF-α; Interleukin-6, IL-6; Macrophage Inflammatory Protein-2, MIP-2) were measured. The oxidative potential and the endotoxin content of each PM sample were determined in a DTT- and LAL-assay respectively. Multiple linear regression was used to assess the relationship between the cellular responses and PM characteristics: concentration, site, size fraction, oxidative potential and endotoxin content.ResultsMost PM samples induced a concentration-dependent decrease in MTT-reduction activity and an increase in pro-inflammatory markers with the exception of the urban background and stop & go traffic samples. Fine and qUF samples of traffic locations, characterized by a high concentration of elemental and organic carbon, induced the highest pro-inflammatory activity. The pro-inflammatory response to coarse samples was associated with the endotoxin level, which was found to increase dramatically during a three-day sample concentration procedure in the laboratory. The underground samples, characterized by a high content of transition metals, showed the largest decrease in MTT-reduction activity. PM size fraction was not related to MTT-reduction activity, whereas there was a statistically significant difference in pro-inflammatory activity between Fine and qUF PM. Furthermore, there was a statistically significant negative association between PM oxidative potential and MTT-reduction activity.ConclusionThe response of RAW264.7 cells to ambient PM was markedly different using samples collected at various sites in the Netherlands that differed in their local PM emission sources. Our results are in support of other investigations showing that the chemical composition as well as oxidative potential are determinants of PM induced toxicity in vitro.

Aβ Mediated Diminution of MTT Reduction—An Artefact of Single Cell Culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Aβ) toxicity in different types of single cell culture. To our knowledge, the influence of Aβ on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-42) and oligomeric Aβ (1-40). In contrast to the findings in single cell cultures, none of these Aβ species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Aβ to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Aβ also did not influence the MTT reduction in the respective tissue. Failure of Aβ penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Aβ (1-40), but not by freshly dissolved Aβ (25-35) or fibrillar Aβ (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Aβ species on MTT reduction. Particularly, the differential effect of oligomeric versus other Aβ forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Aβ oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Aβ, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.