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PubMed1995

Effect of nitroblue tetrazolium on NO synthase and motor function of opossum esophagus.

Nitric oxide mediates neuromuscular events in the opossum esophagus. The NADPH diaphorase stain is used to localize nitric oxide synthase-containing enteric neurons. Cells stain by the NADPH diaphorase technique because they reduce nitroblue tetrazolium to the visible formazan. The effects of nitroblue tetrazolium on neuromuscular function and nitric oxide synthase of esophageal muscle were studied. The NADPH diaphorase stain was performed. Nitroblue tetrazolium inhibited lower esophageal sphincter relaxation, abolished the latency gradient of the off response, and inhibited nitric oxide synthase. The NADPH diaphorase technique stained myenteric plexus nerve cell bodies and nerve processes. Nitroblue tetrazolium is not a nonspecific muscle or nerve toxin, as nerve-mediated cholinergic responses, responses to exogenous nitric oxide, and responses to myogenic stimulation were maintained after nitroblue tetrazolium abolished the off response and lower esophageal sphincter relaxation. Nitroblue tetrazolium inhibits nitric oxide-mediated events and nitric oxide synthase. It stains neurons in the esophageal myenteric plexus.

Animals↗

PubMed1992

Oxygen radicals generated by the enzyme xanthine oxidase lyse rat pancreatic islet cells in vitro.

The endothelium-associated enzyme xanthine oxidase is known to generate reactive oxygen intermediates which may damage the surrounding tissue. We investigated whether reactive oxygen intermediates released by xanthine oxidase exert a toxic effect on isolated rat islet cells. The xanthine oxidase (25 mU/ml)/hypoxanthine (0.5 mmol/l) system released reactive oxygen intermediates in vitro as detected by luminol in a chemiluminescence analysing system. The addition of nicotinamide inhibited the release of reactive oxygen intermediates in a dose-dependent manner (50% inhibition at 20 mmol/l). Exposure of islet cells to enzyme generated reactive oxygen intermediates caused lysis of 39% of the cells within 15 h. Monitoring the mitochondrial function of islet cells by the conversion of tetrazolium bromide to its formazan product revealed a significant reduction of the respiratory activity down to 51% of that of the controls by 30 min after the initiation of the xanthine oxidase reaction. Mitochondrial dysfunction preceded plasma membrane damage. The addition of nicotinamide, a radical scavenger and inhibitor of the DNA repair enzyme poly(ADP-ribose) synthetase protected the islet cells from lysis and partially preserved their mitochondrial activity in the presence of reactive oxygen intermediates. We conclude that activation of the endothelial enzyme xanthine oxidase, known to be induced by mediators of immune cells or by episodes of ischaemia and reperfusion causes islet cell damage with subsequent cell death in early phases of pancreatic islet cell destruction.

Animals↗

PubMed1996

The dynamics of local kinetic parameters of glutamate dehydrogenase in rat liver.

Kinetic parameters of glutamate dehydrogenase (GDH, EC 1.4.1.2) for glutamate were determined in periportal and pericentral zones of adult male and female rat liver lobules under normal fed conditions and after starvation for 24 h. GDH activity was measured as formazan production over time against a range of glutamate concentrations in serial cryostat sections using image analysis. Captured gray value images were transformed to absorbance images and local initial velocities (Vini) were calculated. A hyperbolic function was used to describe the relationship between substrate concentration and local Vini. Under fed conditions, Vmax values were similar in male and female rats (8 +/- 2 and 16 +/- 2 mumol min-1 cm-3 liver tissue in periportal and pericentral zones, respectively). Starvation increased Vmax, especially in pericentral zones of females (to 27 +/- 1 mumol min-1 cm-3 liver tissue). Under fed conditions, the affinity of GDH for glutamate was similar in male and female rats (2.5 +/- 0.5 mM and 3.5 +/- 0.8 mM in periportal and pericentral zones, respectively). Starvation had no effect on K(m) values in male rats, but in female rats affinity for glutamate decreased significantly in both zones (K(m) values of 4.0 +/- 0.1 mM and 8.6 +/- 0.8 mM, respectively). These local changes in the kinetic parameters of GDH indicate that conversion of glutamate to alpha-oxoglutarate cannot be predicted on the basis of GDH concentrations or zero-order activity in the different zones of liver lobules alone.

Animals↗

PubMed1980

Visualization of cellular aggregates cultured on a three dimensional collagen sponge matrix.

A one-step vital stain is described for the macroscopic visualization of histotypic cell aggregates in fetal rat lung organotypic cultures. Organotypic cultures are incubated in 0.05%-0.1% 2,3,5'-triphenyl tetrazolium chloride (TTC) in culture medium (37 degrees C). Living cells reduce the tetrazole to a water-insoluble red colored formazan. Cell aggregates appear as densely stained foci against the lighter background of the Gelfoam substrate. Stained cultures may be scanned macroscopically to determine the degree of reaggregation and assess cell viability. Identification of aggregates by TTC staining improves the efficiency of tissue processing for electron microscopy and does not alter the ultrastructural appearance of the cultured cells.

Animals↗

PubMed1993

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles of Saccharomyces cerevisiae.

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures of Saccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0 degrees C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits features envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.

Cell Membrane Permeability↗

PubMed2001

Comparison of two rapid colorimetric methods for determining resistance of mycobacterium tuberculosis to rifampin, isoniazid, and streptomycin in liquid medium.

The usefulness of two colorimetric methods for the determination of the susceptibility or resistance of Mycobacterium tuberculosis to rifampin, streptomycin, and isoniazid in liquid medium based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was investigated. The agar proportion method was used as the reference method. Results obtained indicate that the sensitivity of the XTT reduction assay for the detection of rifampin resistance was comparable to that observed, and previously described, for the MTT assay. However, the reduction of XTT yields a water-soluble formazan that can be easily quantified without performing additional steps such as addition of lysing buffer and solubilization. Furthermore, the colorimetric assays, based on the reduction of XTT and MTT for the detection of isoniazid and streptomycin resistance in Mycobacterium tuberculosis, were standardized. The inhibition of MTT and

Antibiotics, Antitubercular↗

PubMed1999

Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin.

Depending on the availability of suitable reducing agents, alloxan can be either a prooxidant or an antioxidant. Alloxan and its reduced derivative, dialuric acid, act as a redox couple, driven by reduced glutathione (GSH) or L-cysteine, generating in vitro in the presence of oxygen, both superoxide radical and hydrogen peroxide. The production of superoxide radicals was shown by the appearance of lucigenin chemiluminescence (CL) as well as by the generation of formazan from nitroblue tetrazolium (NBT). The lucigenin CL as well as the NBT reduction was inhibited by superoxide dismutase and partially by catalase. Melatonin inhibited alloxan-mediated CL. In contrast, in the absence of reducing agents, alloxan is a scavenger of superoxide radicals formed by other reactions. Because of the high content of reducing compounds in the cell (e.g. glutathione), it is suggested that alloxan acts in vivo mainly as a generator of reactive oxygen species.

Alloxan↗

PubMed2003

Effects of protein kinase C inhibition and activation on proliferation and apoptosis of bovine retinal pericytes.

AIMS/HYPOTHESIS: Drop-out of capillary pericytes occurs early and selectively in diabetic retinopathy. High glucose concentrations decrease replication and increase apoptosis of cultured pericytes. Since glucose activates protein kinase C, we investigated the effects of modulating this intracellular mediator on replication, cell cycle and apoptosis of cultured bovine retinal pericytes. METHODS: Pericytes cultured in 5.6 or 28 mmol/l glucose were exposed to a protein kinase C activator (phorbol 12-myristate 13-acetate) and/or a selective inhibitor of its beta2 isoform (LY379196). Cells were counted after 7 days. Proliferation by the tetrazolium to formazan assay and DNA synthesis by 5-bromo-2'-deoxyuridine incorporation were measured at day 4. Cell cycle by flow cytometry and apoptosis by ELISA were assessed at day 2. RESULTS: High glucose reduced pericyte replication and increased apoptosis. Protein kinase C activation increased proliferation, while inhibition of its beta2 isoform decreased it. Cell cycle was accelerated by protein kinase C activation and delayed by inhibition. Apoptosis was enhanced by protein kinase C inhibition and reduced by activation. CONCLUSIONS/INTERPRETATION: Protein kinase C inhibition amplifies the anti-proliferative and pro-apoptotic effects of high glucose on cultured pericytes, whereas stimulation reduces apoptosis and promotes proliferation both in physiological glucose and high glucose. Protein kinase C inhibition, proposed for the treatment of diabetic macular edema and proliferative retinopathy, might accelerate pericyte dropout in earlier stages when these cells are still present in retinal capillaries.

Animals↗

PubMed2005

Fatty acid-induced differential regulation of the genes encoding peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta in human skeletal muscle cells that have been differentiated in vitro.

AIMS/HYPOTHESIS: The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) enhances metabolically relevant pathways, such as gluconeogenesis, fatty acid oxidation, thermogenesis, oxidative phosphorylation and mitochondrial biogenesis. Since regulation of the expression of the gene encoding PGC-1alpha (PPARGC1A) by nutrients/metabolites has not been assessed in detail, the aim of this study was to determine whether PPARGC1A (and PPARGC1B) expression is modulated by common plasma fatty acids in human skeletal muscle cells. METHODS: Human myotubes that had been differentiated in vitro were treated with 0.5 mmol/l myristate (C14:0), palmitate (C16:0), stearate (C18:0), palmitoleate (C16:1omega7), oleate (C18:1omega9) or linoleate (C18:2omega6). PPARGC1A/B mRNA was quantified by RT-PCR. Mitochondrial activity was determined by formazan formation. RESULTS: Untreated cells expressed 28-fold more PPARGC1B mRNA than PPARGC1A mRNA (13.33+/-2.86 vs 0.47+/-0.08 fg/mug total RNA, n=5). PPARGC1A expression was increased two- to three-fold by all unsaturated fatty acids (UFAs) tested (p<0.05 each, n=5). In contrast, saturated fatty acids (SFAs) did not modulate PPARGC1A expression. Furthermore, the effect of linoleate was not blunted by palmitate. PPARGC1B mRNA expression was not increased by either the UFAs or the SFAs. SFAs reduced PPARGC1B expression (p<0.05 for palmitate and stearate, n=5). Notably, linoleate reversed palmitate's repressive effect on PPARGC1B. Myotube mitochondrial activity was increased by all UFAs (p<0.01 each, n=5), but was impaired by the SFA stearate (p<0.001, n=5). CONCLUSIONS/INTERPRETATION: We report here that fatty acids differentially regulated expression of the genes encoding the PGC-1 isoforms. Since these effects were accompanied by significant changes in mitochondrial activity, we suggest that the fatty acid-induced regulation of expression of these genes plays an important role in muscle oxidative metabolism.

Carrier Proteins↗

PubMed1998

The effect of organ preservation solutions on kidney tubular and endothelial cells.

Organ preservation solutions have primarily been tested in whole organ animal models. In the current study, we have examined the effect of commonly used organ preservation solutions on both kidney tubular and endothelial cells. Primary human endothelial and kidney tubular cells were incubated at 4 degrees C in the following solutions: 0.9% saline (NS), EuroCollins (EC). University of Wisconsin (UW), or Hank's balanced salts with 5% polyethylene glycol (PEG). Cell viability was assessed by colorometric measurement of mitochondrial reduction of 3 (4,5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT) to purple 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan. After hypothermic storage, cells were incubated at 37 degrees C in media with MTT, and the amount of reduced formazan present was quantified. Endothelial cells preserved in PEG displayed the best viability (P < 0.05). UW provided better cellular viability than EC or NS (P < 0.05). Control endothelial cells preserved in culture media at 37 degrees C displayed the highest absorbance values (P < 0.01). For kidney tubular cells, UW and PEG provided the best cellular protection (P < 0.05). Control kidney tubular cells cultured in complete media at 37 degrees C displayed the highest absorbance values (P < 0.01). Although the model presented here was not part of a truly morphological study, it may be more reliable for the rapid assessment of preservation-induced cell injury than models presented in previous morphological studies and may help in the development of improved preservation techniques.

Cells, Cultured↗

PubMed1995

Influence of glucose on the toxicity of oxophenylarsine in MDCK cells.

Trivalent arsenicals like oxophenylarsine (PhAsO) inhibit cellular pyruvate dehydrogenase, thus leading to a drop of acetylCoA formation and a slow-down of the citric acid cycle. Glucose may protect cells from arsenic toxicity, because increased glycolysis may prevent fatal shortage of ATP. On the other hand, PhAsO has been shown to inhibit glucose uptake in Madin-Darby canine kidney (MDCK) cells. We have investigated the effect of PhAsO on viability, ATP levels and glucose uptake of MDCK cells in the presence of normal (5 mmol/l) and low (0.01 mmol/l) glucose concentrations. At normal as well as at low glucose concentrations, cell viability as assessed by formazan formation was not affected by PhAsO concentrations up to 2 mumol/l within 3 h of observation. At higher PhAsO concentrations viability was diminished earlier and more pronounced in the presence of low glucose concentrations. 10 mumol/l PhAsO induced a drastic drop of ATP within 30 min which was followed by an almost complete loss of viable cells after 180 min in the presence of low glucose concentrations, while at normal glucose levels no influence on ATP contents or on cell viability was detected within 60 min of incubation. On the other hand, glucose uptake, determined as 14C accumulation by cells incubated for 10 min with D-[6-14C]-glucose, was inhibited by PhAsO at low as well as at normal glucose concentrations in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl Coenzyme A↗

PubMed1996

Selective cytotoxicity associated with in vitro exposure of fresh rat renal fragments and continuous cell lines to atractyloside.

The consumption of plants containing the diterpenoid atractyloside (ATR) causes selective proximal tubule injury, renal failure and death in humans. We have compared the effects of ATR in freshly isolated renal proximal tubules and glomeruli from rat and also in cell lines: NRK, derived from the proximal tubules, and MDBK and MDCK more closely representing the distal nephron. The effects of ATR (10-500 microM) on proximal tubules and glomeruli were assessed by changes in lipid peroxidation, de novo protein synthesis and the leakage of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH) and N-acetyl-beta-D-glucosaminidase (NAG). The susceptibility of NRK, MDBK and MDCK cell lines to ATR was assessed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, measuring mitochondrial reduction. Enzyme leakage was the most sensitive of the markers of cell injury in fresh fragments and ranked LDH > GDH > ALP > NAG in proximal tubules. As little as 20 microM ATR caused significant enzyme leakage from proximal tubules, but there were no increases in enzyme leakage from glomeruli at concentrations < and = 500 microM ATR. De novo protein synthesis was only inhibited 50% at ATR concentration > 5 mM in the proximal tubules, but there were no effects in glomeruli. Malondialdehyde production was significantly elevated at 1 mM ATR for proximal tubules, and 500 microM for glomeruli. NRK cells were sensitive to ATR (IC50, 120 microM), but MDBK or MDCK cells were unaffected by < and = 1 mM of this diterpenoid. Both freshly isolated fragments and continuous cell lines representing the proximal tubules are more sensitive to ATR than either glomeruli or cells representing the distal nephron. These data also show that protein synthesis is a less specific and sensitive measure of ATR cytotoxicity than enzyme leakage in fragments. MTT reduction to formazan was the most sensitive in the NRK cell line. The low levels of lipid peroxidation products in proximal tubular fragments or sensitive renal cell lines at toxic levels of ATR suggest that oxidative injury is not a key mechanism.

Animals↗

PubMed1999

Estimation of PC12 cell numbers with acid phosphatase assay and mitochondrial dehydrogenase assay: dopamine interferes with assay based on tetrazolium.

Both the acid-phosphatase and mitochondrial dehydrogenase assay have been used to quantify cell numbers. The commonly used acid-phosphatase assay uses p-nitrophenyl phosphate as a substrate, while the mitochondrial dehydrogenase assay is based on the conversion of tetrazolium to formazan. Our experimental results showed that the former assay was more sensitive in detecting small numbers of PC12 cells (200-10,000 cells/well), whereas the latter was useful for larger numbers of cells (2000-40,000 cells/well). The number of PC12 cells decreased after dopamine treatment, according to the acid-phosphatase assay and by direct cell counts under a light microscope. However, the optical densities measured by the mitochondrial dehydrogenase assay increased after dopamine treatment. We tried to clarify discrepancies between the two assays, since dopamine is an important neurotransmitter and both assays are commonly used to estimate cell numbers. To elucidate the interference between dopamine and tetrazolium salt, cell-free control experiments were performed. Dopamine and other catecholamines (adrenaline and noradrenaline) reacted with tetrazolium and, thus, produced a false positive reaction in the assay. We therefore conclude that the tetrazolium assay is not a suitable method for evaluating the number of catecholamine-treated cells, while the acid-phosphatase assay is reliable and sensitive.

Acid Phosphatase↗

PubMed2001

Immunocytochemical distribution of nitric oxide synthase in the human corpus cavernosum: an electron microscopical study using the tyramide signal amplification technique.

Nitric oxide has proven to be an important mediator in the relaxation of human cavernosal smooth muscle. Nevertheless, there are many inconsistencies in the literature regarding the cellular and subcellular distribution of endothelial nitric oxide synthase in the human penis. The purpose of this study was to reexamine the localization of eNOS and nNOS in the cellular anatomy of the human cavernous body by means of electron microscopical immunocytochemistry in combination with the tyramide signal amplification technique (TSA). Using specific antibodies against eNOS and nNOS, the NAPDH-diaphorase reaction and advanced protocols for fixation and staining procederes, the occurrence of NOS isoenzymes eNOS and nNOS were examined in cavernosal specimens of ten male patients who were subjected to surgery for penile deviation. eNOS immunoreactivity and NADPH-d staining was seen to be significantly present in the endothelial cells covering the cavernous spaces and in the endothelium of helicine arteries. In endothelial cells, the NADPH-d reaction product BSPT-formazan was abundantly detectable attached to membranes of the endoplasmatic reticulum and the mitochondria whereas posititve eNOS immunostaining was seen in the endothelial cells throughout their cytoplasm without any particular relation to organelles. No considerable eNOS immunoreactivity was detectable in the trabecular smooth muscle cells. nNOS staining was found in nerve fibers innervating the cavernous body and cavernosal arteries. Our results counteract the hypothesis of the cavernous smooth muscle as a local source of NO and underline the importance of an intact endothelial function for penile erection and the contribution of eNOS to this process.

Biotin↗

PubMed2003

Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal lymphocytes. Phenothiazines and leukemia.

PURPOSE: The purpose of the present study was to investigate the effects of phenothiazines (at clinically relevant doses) on the viability and proliferation of leukemic cell lines and normal lymphocytes, and to investigate the possibility of specific induction of apoptosis in leukemic cells. METHODS: Phenothiazines with different chemical structure and hydrophobicity were used: chlorpromazine (CPZ); levomepromazine (LVPZ); prometazine (PMZ); trifluoperazine (TFPZ); thioridazine (TRDZ). The leukemic cell lines used were: Daudi and Raji (derived from Burkitt's lymphoma), K-562 (derived from myelogenous leukemia), and BALL-1, MOLT-4, HPB-ALL and CCRF-HSB-2 (derived from acute lymphoblastic leukemia). The cytotoxicity of the phenothiazines was determined by a CellTiter-Glo luminescent cell viability assay, using ATP bioluminescence as a marker of cell viability as well as a marker of mitochondrial activity. The proliferation of leukemic cells was determined using a CellTiter-AQ cell proliferation assay which is based on the reduction of a methyl-tetrazolium compound to the formazan product. Apoptosis induction was estimated using phosphatidylserine (PSer) translocation to the cell surface and DNA fragmentation as characteristics of the process. RESULTS: Phenothiazines (at concentrations in the range 0.1-10 micro M) did not affect the viability of normal lymphocytes during a 24-h incubation. Moreover, about 15-20% increase in ATP bioluminescence was observed in normal cells during treatment with 40 micro M phenothiazines. In contrast, the phenothiazines manifested strong cytotoxicity and antiproliferative activity against leukemic cells. The most powerful drugs were TFPZ and TRDZ, followed by CPZ. They showed a significant cytotoxic effect against leukemic cells even at 5-10 micro M. The most sensitive cell lines were MOLT-4 and Raji, and the most resistant were HPB-ALL and CCRF-HSB-2. All phenothiazines induced PSer exposure on the surface of leukemic cells, but not of normal lymphocytes. TFPZ, TRDZ and CPZ also induced DNA fragmentation in almost all leukemic cell lines during a 48-h incubation. The strongest apoptotic agent was TRDZ. The apoptosis induction was not accompanied by a significant release of cytochrome c from the mitochondria into the cytoplasm of native cells. Moreover, the drugs markedly suppressed Ca(2+)-induced cytochrome c release in isolated mitochondria of leukemic cells. CONCLUSIONS: The results suggest that in clinically relevant doses (up to 20 micro M) some phenothiazines (TFPZ, TRDZ, CPZ) expressed a selective cytotoxicity and antiproliferative activity, and induced apoptosis in leukemic cells without any influence on the viability of normal lymphocytes. It is considered that the mechanism of apoptosis induction in phenothiazine-treated leukemic cells is associated with inhibition of mitochondrial DNA polymerase and decreased ATP production, which are crucial events for the viability of cancer cells.

Adenosine Triphosphate↗

PubMed2006

Exposure to carbon dioxide and helium reduces in vitro proliferation of pediatric tumor cells.

BACKGROUND: Minimally invasive techniques are increasingly applied to children with malignant tumors. We showed previously that CO(2) used for pneumoperitoneum modulates the function of macrophages and polymorphonuclear cells via direct effects and via acidification. Numerous in vitro and small animal model studies also confirmed an alteration of the behavior of several types of adult tumor cells by CO(2). The impact of CO(2) and other gases used for pneumoperitoneum on the behavior of various pediatric tumors has not yet been determined. METHODS: Cell lines of neuroblastoma (IMR 32, SK-N-SH, Sy5y), lymphoma (Daudi), hepatoblastoma (Huh 6), hepatocellular carcinoma (Hep G2), and rhabdomyosarcoma (Te 671) were incubated for 2 h. Incubation was performed with 100% CO(2), 100% helium, and 5% CO(2) as control. Cell proliferation was determined by the MTT-assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by actively growing cells to produce a blue formazan product. The MTT-assay was performed before, directly after incubation, and daily for 4 days. Vitality of the cells was determined by trypan blue. The extracellular pH during incubation was measured during gas exposition every 10 min using Bayer Rapid Lab 855. RESULTS: CO(2) for 2 h significantly decreased the proliferation of neuroblastoma, lymphoma, hepatoblastoma, and hepatocellular carcinoma cells. This decrease persisted over 4 days in neuroblastoma, lymphoma, and hepatocellular carcinoma cells. The CO(2) had no impact on hepatoblastoma and rhabdomyosarcoma cells. Helium had a similar effect on neuroblastoma cells. After 4 days, a significant decrease of cell activity was found in two neuroblastoma cell lines and in hepatoblastoma cells. Helium had no effect on lymphoma and hepatocellular carcinoma cells. The extracellular pH was 6.2 during incubation with CO(2), and 7.6 during incubation with helium. CONCLUSION: CO(2) and helium may affect the proliferation of some pediatric tumor cell lines in vitro. However, some of these effects and the impact on the extracellular pH are differential. The role of pH modulation, hypoxia and direct effects of gases remain to be investigated before a general recommendation on the use of minimally invasive techniques in pediatric oncology can be given.

Carbon Dioxide↗

PubMed1998

Effect of low-power red light laser irradiation on the viability of human skin fibroblast.

Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm2). The energy densities were 2 J/cm2 and 12 J/cm2, respectively, and the irradiation was carried out at a temperature of 22 degrees C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37 degrees C, in the presence of 5% CO2, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation.

Cell Survival↗

PubMed2006

Oregano (Lippia spp.) kills Giardia intestinalis trophozoites in vitro: antigiardiasic activity and ultrastructural damage.

In the world, giardiosis is still a very important parasitic disease; only in Asia, Africa and America, there are more than 200 million of infected people in a year. The usual treatments are drugs that produce undesirable secondary effects, perhaps favouring the resistant strain selection. One alternative is to research compounds from plants used as antidiarrhoeic or antiparasitic in the traditional medicine. In a previous work, we found that Lippia beriandieri (Oregano) revealed to be more potent than tinidazole, a common antigiardiasic drug. In this current work, we tested the cell viability by re-culture and reduction of MTT-tetrazolium salts to MTT-formazan, and we showed the effect of oregano ethanolic extracts against Giardia intestinalis (synonyms: Giardia duodenalis, Giardia lamblia) trophozoites at concentrations ranging form 58 to 588 microg. We demonstrated the ultrastructural injury produced by oregano extracts in this parasite. Trophozoites lost their size and shape and showed damage in nucleus structure, perhaps by breaking the pattern of nucleoskeleton proteins.

Animals↗