Intracellular Dehydrogenase Activity Research Articles

The effect of repeated passaging on the susceptibility of human proximal tubular HK-2 cells to toxic compounds.

The human proximal tubular HK-2 cell line is an immortalized cell line commonly used for studying proximal tubular toxicity. Even as their use is presently increasing, there unfortunately are no studies focused on functional changes in HK-2 cells associated with passaging. The aim of the present study, therefore, was to evaluate the functional stability of HK-2 cells during 13 weeks of continuous passaging after 6 and 24 h of treatment with model nephrotoxic compounds (i.e., acetaminophen, cisplatin, CdCl(2)). Short tandem repeat profile, the doubling time, cell diameter, glutathione concentration, and intracellular dehydrogenase activity were measured in HK-2 cells at each tested passage. The results showed that HK-2 cells exhibit stable morphology, cell size, and cell renewal during passaging. Mean doubling time was determined to be 54 h. On the other hand, we observed a significant effect of passaging on the susceptibility of HK-2 cells to toxic compounds. The largest difference in results was found in both cadmium and cisplatin treated cells across passages. We conclude that the outcomes of scientific studies on HK-2 cells can be affected by the number of passages even after medium-term cultivation and passaging for 13 weeks.

Transient increase in cellular dehydrogenase activity after cadmium treatment precedes enhanced production of reactive oxygen species in human proximal tubular kidney cells.

Cadmium is a heavy metal causing toxicity especially in kidney cells. The toxicity is linked also with enhanced oxidative stress leading to cell death. On the other hand, our recent experiments have shown that an increase of total intracellular dehydrogenases activity can also occur in kidney cells before declining until cell death. The aim of the present study, therefore, was to evaluate this transient enhancement in cell viability after cadmium treatment. The human kidney HK-2 cell line was treated with CdCl(2) at concentrations 0-200 microM for 2-24 h and intracellular dehydrogenase activity was tested. In addition, we measured reactive oxygen species (ROS) production, glutathione levels, mitochondrial membrane potential, and C-Jun-N-terminal kinase (JNK) activation. We found that significantly increased dehydrogenase activity could occur in cells treated with 25, 100, and 200 microM CdCl(2). Moreover, the results showed an increase in ROS production linked with JNK activation following the enhancement of dehydrogenase activity. Other tests detected no relationship with the increased in intracellular dehydrogenase activity. Hence, the transient increase in dehydrogenase activity in HK-2 cells preceded the enhancement of ROS production and our finding provides new evidence in cadmium kidney toxicity.

Adaptation response of Pseudomonas fragi on refrigerated solid matrix to a moderate electric field

BackgroundModerate electric field (MEF) technology is a promising food preservation strategy since it relies on physical properties—rather than chemical additives—to preserve solid cellular foods during storage. However, the effectiveness of long-term MEF exposure on the psychrotrophic microorganisms responsible for the food spoilage at cool temperatures remains unclear.ResultsThe spoilage-associated psychrotroph Pseudomonas fragi MC16 was obtained from pork samples stored at 7 °C. Continuous MEF treatment attenuated growth and resulted in subsequent adaptation of M16 cultured on nutrient agar plates at 7 °C, compared to the control cultures, as determined by biomass analysis and plating procedures. Moreover, intracellular dehydrogenase activity and ATP levels also indicated an initial effect of MEF treatment followed by cellular recovery, and extracellular β-galactosidase activity assays indicated no obvious changes in cell membrane permeability. Furthermore, microscopic observations using scanning and transmission electron microscopy revealed that MEF induced sublethal cellular injury during early treatment stages, but no notable changes in morphology or cytology on subsequent days.ConclusionOur study provides direct evidence that psychrotrophic P. fragi MC16 cultured on nutrient agar plates at 7 °C are capable of adapting to MEF treatment.

Agrowaste to vanillin conversion by a natural Pediococcus acidilactici strain BD16

ABSTRACTOwing to its flavoring, antimicrobial, antioxidant and anticarcinogenic nature, vanillin is widely used in foods, beverages, perfumes and pharmaceutical products. Ferulic acid (FA) is an important precursor of vanillin which is abundant in cereals like maize, rice and wheat and sugar beet. A major drawback of microbial vanillin production from FA is the degradation and biotransformation of toxic vanillin to other phenolic derivatives. The present study is undertaken to explore microbial vanillin production from FA precursor rice bran by employing vanillin-resistant Pediococcus acidilactici BD16, a natural lactic acid bacteria isolate. Extracellular, intracellular and cellular vanillin dehydrogenase activity was found least, which was minimized vanillin degradation, and the strain resists more than 5 g L−1 vanillin in the medium. A metabolomics approach was followed for the detection of FA, vanillin and other metabolites generated during fermentation of rice bran medium. A metabolic pathway was also predicted for vanillin biosynthesis. Approximately 1.06 g L−1 of crude vanillin was recovered from rice-bran-containing medium and this further offers scope for the industrial utilization of the organism and its genetic manipulation to enhance production of biovanillin.

Electrical Retrieval of Living Microorganisms from Cryopreserved Marine Sponges Using a Potential-Controlled Electrode.

The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at −80 °C. A −0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms.

Intestinal safety of water-soluble beta-cyclodextrins in paediatric oral solutions of spironolactone: effects on human intestinal epithelial Caco-2 cells.

Effects of water-soluble beta-cyclodextrins (beta CDs) on intestinal epithelial integrity were investigated, to establish the safe use of these beta CDs as solubilizers of spironolactone in paediatric enteral solutions. Mannitol permeability and transepithelial resistance (TER) of human intestinal epithelial Caco-2 cell monolayers during exposure to dimethyl-beta-cyclodextrin (DM beta CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) and sulphobutyl ether beta-cyclodextrin (SBE beta CD) were followed. Staining methods were used to discern cells with damaged membranes and to study the integrity of cytoskeletal actin and tight junctions. Cytotoxicity of the beta CDs was tested by effects on intracellular dehydrogenase activity. Exposure to HP beta CD and SBE beta CD solutions had only minor effects on the integrity of Caco-2 cell monolayers. In contrast, DM beta CD clearly increased the epithelial permeability for the hydrophilic marker [14C]mannitol across Caco-2 monolayers, decreased TER and showed a dose-dependent cytotoxicity. According to staining, DM beta CD increased the permeability of the apical cell membrane without discernable effects on cytoskeletal actin. HP beta CD and SBE beta CD appear to be safe additives for use in enteral spironolactone preparations with respect to their acute local effects on epithelial integrity.

Trial to evaluate effects of ambient particulate matter on health: A preliminary study using two-dimensional gel electrophoresis

Particulate air pollution is a serious problem all over the world, and the development of a method to evaluate the health effects of ambient particles is necessary. In this study, cells cultured in vitro were exposed to particles sampled at the side of a main road, and their protein expression levels were examined. Ambient particles were collected at the side of a main road using a high-volume air sampler. Some of the collected particles (crude particles) were treated with an organic solvent to remove chemical components, and the resulting residues were used as residual particles. Cells from the mouse alveolar epithelial cell line LA-4 were inoculated into tissue-culture dishes at 1.4×10(4)/cm(2), exposed to each type of particle or artificial carbon particles (Printex 90) that were dispersed using an ultrasonic homogenizer by mixing in the medium twice at 24 and 48 hours, and incubated for up to 72 hours after the start of inoculation. After exposure, the number of cells and intracellular dehydrogenase activity were measured. Proteins extracted from the cells were subjected to two-dimensional gel electrophoresis with isoelectric focusing at pHs 4-7 using a 10% acrylamide gel, and their expression levels were analyzed after fluorescent staining. The intracellular dehydrogenase activity of the cells significantly decreased as a result of exposure to the residual (0.70-fold) and crude (0.84-fold) particles compared with that of the control, but it showed no change as a result of exposure to Printex 90. The protein expression levels in the cells exposed to the particles increased or decreased similarly, but different expression levels were also observed. There were differences in the effects observed between the cells exposed to the artificial carbon particles and those exposed to particles collected from ambient air. This study indicates that protein expression levels in cells change in response to exposure to particles collected from ambient air. To evaluate the effects of particles on health, it is considered necessary to use particles collected from ambient air.

Gene Expression Profiles in Mouse Lung Tissue after Administration of Two Cationic Polymers Used for Nonviral Gene Delivery

This study compared gene expression profiles in mouse lungs after administration of the cationic polymers polyethyleneimine (PEI) or chitosan alone or formulated with a luciferase reporter plasmid (PEI-pLuc, chitosan-pLuc). The polymers and formulations were administered intratracheally to Balb/c mice at doses judged to be nontoxic according to intracellular dehydrogenase activity and tissue morphology. RNA was isolated from the lungs 24 or 72 h after administration, and a dedicated stress and toxicology cDNA array was used to monitor the in vivo response to the gene delivery system in the lung tissue. The gene expression profiles differed between the PEI and chitosan groups with regard to both the total number and the type of expressed genes. Chitosan-pLuc upregulated genes that protect the cell from oxidative stress and inflammation, such as heme oxygenase-1 and catalase, whereas PEI-pLuc upregulated genes involved in inflammatory processes, such as the cyclooxygenases 1 and 2, indicating possible involvement in the development of adverse reactions. However, both polymers activated genes involved in reaction to stress, such as DNA damage repair. Furthermore, in the PEI group, chaperone genes and members of the p38 mitogen-activated protein kinase pathway were also upregulated, suggesting a possible explanation for the better performance of PEI in gene delivery systems. The results indicate that gene expression profiling is a useful and sensitive tool for the evaluation of tissue responses after administration of polymers or gene delivery systems. The results also suggest a possible explanation for the differences in gene delivery performance between the two polymers in gene delivery systems.

Bioremediation of Petroleum Contaminated Beach Sediments: Use of Crude Palm Oil and Fatty Acids to Enhance Indigenous Biodegradation

Amendment of simple organic carbon, in the presence of inorganic nutrients, to oil contaminated beach sediments can potentially stimulate the biodegradation of hydrocarbons by the indigenous microbial biomass. The ability of crude palm oil (CPO) and fatty acids, in the presence of soluble inorganic nutrients (C:N:P = 100:10:1), to stimulate biodegradation in sediments amended with Arabian light crude oil was investigated in laboratory microcosms over a 30-day period. The presence of nutrients alone enhanced the most probable number (MPN) of hydrocarbon degrading bacteria by almost 12 times after 30 days, and by 26 times in sediment also amended with CPO, thereby confirming its ability to stimulate the biomass. Addition of individual fatty acids to nutrients-amended sediments resulted in MPN enhancements of up to 170 times, where straight alkanes (i.e., C17 and C18) were completely degraded. Amendment with CPO, myristic, oleic, linoleic and palmitic acids (0.5% dry weight equivalent) also enhanced the metabolic activity of the entire microbial biomass, as measured by intracellular dehydrogenase activity, reaching a maximum of 30 μg INTF g dry sed−1 h−1. Loss of branched alkanes was significantly enhanced, where loss of pristane was complete and phytane loss reached 98% in sediment treated with myristic acid. This study highlights the considerable benefit of adding readily oxidizable fatty acids for enhancing in situ bioremediation of hydrocarbons in nutrient-amended beach sediments.

Cryopreservation of alginate-encapsulated recombinant cells for antiangiogenic therapy.

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to -7.5 degrees C and nucleated. Thereafter, the samples were cooled at a rate of 0.25 degrees C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Apoptosis of Neuro2a cells induced by lysosphingolipids with naturally occurring stereochemical configurations

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [3H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intra-nucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40–80 μM of lysosphingolipids for 24–48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37 °C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation. These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses. —Sueyoshi, N., T. Maehara, and M. Ito. Apoptosis of Neuro2a cells induced by lysosphingolipids with naturally occurring stereochemical configurations.

Chitosans as absorption enhancers for poorly absorbable drugs. 1: Influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (Caco-2) cells.

Chitosan has recently been demonstrated to effectively enhance the absorption of hydrophilic drugs such as peptides and proteins across nasal and intestinal epithelia (1-3). In this study, the effect of the chemical composition and molecular weight of chitosans on epithelial permeability and toxicity was investigated using monolayers of human intestinal epithelial Caco-2 cells as a model epithelium. Eight chitosans varying in degree of acetylation (DA) and molecular weight were studied. The incompletely absorbed hydrophilic marker molecule 14C-mannitol was used as a model drug to assess absorption enhancement. Changes in intracellular dehydrogenase activity and cellular morphology were used to assess toxicity. Chitosans with a low DA (1 and 15%) were active as absorption enhancers at low and high molecular weights. However, these chitosans displayed a clear dose-dependent toxicity. Chitosans with DAs of 35 and 49% enhanced the transport of 14C-mannitol at high molecular weights only, with low toxicity. One chitosan (DA = 35%; MW = 170 kD) was found to have especially advantageous properties such as an early onset of action, very low toxicity, and a flat dose-absorption enhancement response relationship. The structural features of chitosans determining absorption enhancement are not correlated with those determining toxicity, which makes it possible to select chitosans with maximal effect on absorption and minimal toxicity.

Determination of intracellular dehydrogenase activities using flow injection analysis

Reproducible rapid methods for cell disintegration in miniaturized flow cells were developed. The disintegration of yeast cells by ultrasonics and ultraturrax in a miniaturized flow through chamber was compared with the mixer mill disintegration. The flow through chamber was combined with a sterile on-line sampling device to take samples from yeast cell suspensions and with an automated flow injection analysis (FIA) set-up, to determine the released enzyme activities. The FIA set-up was tested with the sequential determination of malate dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase in mixtures. The automated FIA determination of the intracellular formate dehydrogenase of yeast cells ( Hansenula polymorpha) was performed.

Toxicological and absorption enhancing effects of glycofurol 75 and sodium glycocholate in monolayers of human intestinal epithelial (Caco-2) cells

The effects of glycofurol 75 (GF) and sodium glycocholate (GC) on the toxicity and permeability of the human intestinal cell line Caco-2 were studied. The intracellular dehydrogenase activity of the cells was used as a measure of the toxicity. Concentrations of GC from approx. 10 mM inhibited the intracellular dehydrogenase activity and above 40 mM the activity was less than 10% of the initial level. The concentrations resulting in 50% inhibition (IC 50) were 24.2 mM (1.2%) and 380 mM (6.8%) for GC and GF respectively. GF concentrations of less than 100 mM did not affect the activity. The effects of GF and GC on the absorption of the hydrophilic marker molecule [ 14C]mannitol were studied at concentration levels corresponding to no (23.7 mM GF and 5.1 mM GC), about 25% (117 mM GC and 17.2 mM GC) and 50% (380 mM GF and 24.2 mM GC) inhibition in dehydrogenase activity. The apparent permeability coefficient for mannitol in control monolayers was 5.7 × 10 −8 cm/s. 5.1 mM GC did not enhance the permeability, whereas 17.2 and 24.2 mM enhanced it significantly ( p<0.001). GF (380 mM) did not enhance the permeability. The apparent permeability coefficient of insulin in control monolayers was ≦ 9.8 × 10 −8 cm/s, but varied considerably. 24.2 mM GC enhanced the P app significantly ( p<0.001), whereas GF (380 mM) did not affect the absorption of insulin. The Caco-2 cells were studied by transmission electron microscopy after exposure to 380 mM GF for 1 h. Cells exposed to a mannitol solution of the same osmolality as 380 mM GF (about 700 mosm) and untreated monolayers of Caco-2 cells served as controls. GF caused morphological alteration of the epithelial cells resulting in a distorted appearance with distorted appearance with disordered microvilli, disorganized terminal web and intracellular vacuols. This effect is probably due to the high osmolality as the cells exposed to mannitol displayed the same distorted appearance. However, no effect could be observed on the integrity of the apical cell membrane and the tight junctions.

Epithelial Transport of Drugs in Cell Culture. VII: Effects of Pharmaceutical Surfactant Excipients and Bile Acids on Transepithelial Permeability in Monolayers of Human Intestinal Epithelial (Caco‐2) Cells

The effects of anionic (sodium dodecyl sulfate and sodium dioctyl sulfosuccinate) and nonionic (polysorbate 80 and polyoxyl 40 hydrogenated castor oil) synthetic surfactants and bile acids (sodium taurocholate, sodium taurodeoxycholate, and sodium taurodihydrofusidate) on epithelial integrity were studied in monolayers of human intestinal epithelial (Caco‐2) cells grown on microporous polycarbonate filters. The effects of the surfactants on intracellular enzyme activity, cell monolayer permeability, and morphology were studied. The effects on permeability were studied by two methods: measurements of transport of marker molecules (mannitol and polyethylene glycol) and measurements of transepithelial electrical resistance. All surfactants demonstrated concentration‐dependent effects on intracellular enzyme activities, permeability, and morphology. The effects of the anionic surfactants were more pronounced than those of the nonionic surfactants. The effects on transepithelial electrical resistance correlated with intracellular dehydrogenase activity. Fluxes of marker molecules were the most sensitive measure of epithelial integrity. The results indicate that the hydrophilic marker molecules permeate the epithelial monolayers through different pathways at different concentrations of the surfactants. The effects of the surfactants were reversible at intermediate concentrations, even though the morphology of the monolayers had changed. The results agree with published data obtained with experimental animals and indicate that Caco‐2 cells can be used to study the concentration‐dependent effects of surfactants and other pharmaceutical additives on intestinal epithelial permeability.

Evaluation of the kinetics of the intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes measured by amperometric methods

Amperometric methods were used to study the kinetics of intracellular reduction of 2,6-dichlorophenolindophenol (DCIP) in normal and transformed hepatocytes with glucose and succinate as substrates. The curves showing the formation of DCIP red as a function of time were biphasic, the first part obeying the equation of a pseudo-first-order reaction, the final part corresponding to Michaelis-Menten kinetics. A statistical method was used to estimate pseudo-first-order rate constants k as well as K m and V max values. At saturating glucose concentrations k, K m and V max values were higher in normal compared to transformed cells. Decreasing glucose concentrations revealed lowered saturation concentrations in tumour cells compared to normal cells. With succinate as substrate for hepatocytes, k values were higher than with glucose, while K m and V max were about the same. Hepatoma cells did not metabolize succinate. k values could be attributed to intracellular dehydrogenase activities including cytosolic and mitochondrial processes. Differences in pseudo-first-order rate constants between normal and tumour cells may therefore represent characteristic alterations associated with transformation.

Gastric secretion and parietal cells. I. The relationship between the rates of secretion and the intracellular succinic dehydrogenase activity in resected human stomachs.

Gastric secretion and parietal cells. I. The relationship between the rates of secretion and the intracellular succinic dehydrogenase activity in resected human stomachs.

Dehydrogenase activity of rat epiphyseal cartilage.

SummaryUtilizing the tetrazolium technic, intracellular dehydrogenase activity has been localized in epiphyseal cartilage cells. The most favorabe substrates appear to be succinate. isocitrate, citrate, lactate and malate.