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PubMed2001

An iron-regulated ferric reductase associated with the absorption of dietary iron.

The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway.

Amino Acid Sequence↗
PubMed1997

Use of XTT-assay to assess the cytotoxicity of different surfactants and metal salts in human keratinocytes (HaCaT). A feasible method for in vitro testing of skin irritants.

Because of the increasing need of reliable skin irritation tests and in order to reduce the number of animal experiments, in vitro alternatives have to be developed. We studied four surfactants and five metal salts for their cytotoxic potency in HaCaT cells, a spontaneously immortalized human keratinocyte line. The endpoint used to assess cellular viability was metabolization of the tetrazolium salt XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). The tested substances revealed a significant rank order of their cytotoxicity at an exposure time of 24 h. It was 1) benzalkonium chloride, 2) sodium lauryl sulphate, and 3) Tween 20 (polyoxyethylene sorbitanmonolaurate) and Tween 80 (polyoxyethylene sorbitanmonooleate), for the surfactants; and 1) potassium bichromate, 2) copper sulphate, 3) cobalt chloride and palladium chloride, and 4) nickel sulphate, for the metal salts. There is an excellent correlation to the rank order of their known irritative potency in vivo. Being practicable and effective, the presented XTT-assay on HaCaT cells would be well suitable for an initial orientating screening of substances, subsequently followed by irritation tests directly in humans.

Benzalkonium Compounds↗
PubMed2000

[Selective photodynamic destruction of leukemic cells].

BACKGROUND: Residual leukemic cells if present in autologous bone marrow grafts or CD34+ concentrates obtained from peripheral blood may increase the risk of relapse after autotransplantation. We are presenting the employment of a new method which was introduced into the photodynamic therapy, namely enhancement of synthesis of the photosensitizing compound, protoporphyrin IX, in cancer cells, following application of its metabolic precursor, 5-aminolevulinic acid, for the specific destruction of leukemic cells. METHODS AND RESULTS: By determining cell viability using tetrazolium salt reduction (MTT), by flow cytometrypropidium iodide assay and by determining cell proliferation using bromodeoxyuridine incorporation we studied the effect of photodynamic therapy based on the application of 5-aminolevulinic acid on the cells of leukemic cell lines HL60 (human promyelocytic leukemia), HEL (erythroleukemia), DAUDI (B-cell leukemia), JURKAT (T-cell lymphoma), blast cells of patients with acute myelogenous leukemia as well as on normal lymphocytes and normal human bone marrow progenitors. In in vitro experiments photodynamic therapy based on an administration of 5-aminolevulinic acid (1 mM, 4 h, 18 J/cm2) lowered the number of viable leukemic cells by over 2 orders (with the exception of HEL cells) and eliminated blast cells in mononuclear cell preparations of six out of seven patients with acute myelogenous leukemia. On the other hand the viability of normal resting lymphocytes was little affected by photodynamic therapy (number of necrotic cells increased from 6 to 11%) and also the clonogenic activity of the progenitor cells of normal bone marrows did not decrease substantially (CFU-GM to 60% and BFU-E to 55% of the original activity). CONCLUSIONS: Photodynamic therapy based on the application of 5-aminolevulinic acid is a perspective method for the specific destruction of leukemic cells in autologous transplants.

Aminolevulinic Acid↗
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↗
PubMed1979

Improvement of the glucose oxidase immunoenzyme technic. Use of a tetrazolium whose formazan is stable without heavey metal chelation.

Glucose oxidase immunoenzymatic localization provides a simple way to show antigens in mammalian tissues, with no need for the quenching of endogenous nonspecific staining. The method is useful for the demonstration of many antigens in formalin-fixed, paraffin-embedded tissues. Improvement of this technic by the use of p-nitro blue tetrazolium chloride (NBT) as the disclosing reagent provides a stable, finely grained localization not possible with the previously used thiazolyl blue (MTT). The described modification makes available an ideal immunoenzymatic stain for the study of tissues at the level of the light microscope. This stain is especially useful for the examination of tissues with hemorrhagic or inflammatory lesions, since there is no endogenous background staining. Slides are permanent, and the technic can be used with peroxidase-labeled antibody to localize two antigens in the same tissue.

Animals↗
PubMed2003

[Reversal of multidrug resistance in leukemic cell line K562/AO2 by chlordelazine in vitro].

OBJECTIVE: Some recent studies revealed that phenthiazine might be able to reverse tumor cell drug-resistance. Chlorderazin belongs to the phenthiazine compounds. The study aimed to investigate the reversing effect and mechanism of chlorderazin on multidrug resistance of leukemic cell line K562/AO2. METHODS: (1) The cytotoxicities of chlorderazin were assayed with the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. (2) The reverse effect of chlorderazin on K562/AO2 cells was analyzed with MTT method. The multidrug resistance reversal index (RI) was equal to the ratio of control group IC(50)/test group half inhibition concentration IC(50). (3) The intracellular daunorubicin (DNR) concentrations were measured by the flow cytometry. (4) Mdr1 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). The ratio of mdr-1/beta-actin density was calculated. RESULTS: (1) Chlorderazin 3 micro g/ml showed little toxicity to K562/AO2 cells and the suppression rate was less than 5%, so the concentration of 3 micro g/ml chlorderazin was selected as the experiment concentration. (2) The cytotoxicities of DNR to K562/AO2 were enhanced by 3 micro g/ml of chlorderazin (P < 0.05) and RI was 1.901. (3) Chlorderazin of 3 micro g/ml could increase the intracellular DNR accumulation significantly (P < 0.05), and the fluorescence staining by the flow cytometry was higher (250.95 +/- 18.96) than the control group (112.75 +/- 15.78) and shift right in K562/AO2 cells treated with chlorderazin, and the difference was significant (P < 0.05). (4) Chlorderazin has no significant influence to the expression level of mdr-1 mRNA. Both test group and control group showed a clear mdr-1 mRNA band located at the position of 157 kb. The ratios of mdr-1/beta-actin density were 0.414 +/- 0.012 in the test group and 0.447 +/- 0.027 in the control group, respectively, and the difference was not significant (P > 0.05). CONCLUSION: Chlorderazin could reverse the multidrug resistance by increasing the intracellular DNR accumulation in K562/AO2 cells. The effects had no correlation to the mdr-1 gene. Further study is needed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent.

NAD(P)H:quinone oxidoreductase (NQO1; DT-diaphorase) is elevated in certain tumors, such as non-small cell lung cancer (NSCLC). Compounds such as mitomycin C and streptonigrin are efficiently bioactivated by NQO1 and have been used in an enzyme-directed approach to chemotherapy. Previously, 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) was identified as a potential antitumor agent based on its high rate of bioactivation by human NQO1 and its selective cytotoxicity to cells containing elevated NQO1. RH1, a water-soluble analogue of MeDZQ synthesized in this work, was a better substrate for recombinant human NQO1 than the parent compound. RH1 was, correspondingly, more cytotoxic to human tumor cells expressing elevated NQO1 activity (H460 NSCLC and HT29 human colon carcinoma), as measured by 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium assay, than it was to cells deficient in NQO1 activity (H596 NSCLC and BE human colon carcinoma). RH1 exhibited a greater selective toxicity (ratio of IC50s in H596:H460 and BE:HT29) to cells with elevated NQO1 activity relative to MeDZQ. Additionally, we report the establishment of a stable line of BE human colon carcinoma cells transfected with wild-type human NQO1 (BE-NQ7). BE cells are devoid of NQO1 activity due to a homozygous point mutation in the NQO1 gene. In comparison to the parental cell line, RH1, MeDZQ, and mitomycin C were significantly more cytotoxic to BE-NQ7 cells (17-, 7-, and 3-fold, respectively), confirming that the presence of NQO1 is sufficient to increase cytotoxicity of these antitumor quinones. These data suggest that RH1 may be an effective NQO1-directed antitumor agent for the therapy of tumors with elevated NQO1 activity, such as NSCLC.

Antineoplastic Agents↗
PubMed1975

The involvement of superoxide anions in the autoxidation of various cofactors of cysteamine-oxygenase.

The reduction of tetranitroblue tetrazolium with cysteamine, mediated by a number of dyes, elemental sulphur, elemental selenium and selenide, under aerobic conditions, was inhibited to various extent upon addition of superoxide dismutase. A strict parallelism between the ability to produce O2- ions and the property of those compounds to act as cofactors for cysteamine-oxygenase, to yield hypotaurine, has been observed. Based on the fact that the autoxidation of cysteamine also gives rise to O2- formation, though to a minor extent, we propose a mechanism for cysteamine-oxygenase action. This mechanism was derived from the data obtained in the model system studied.

Animals↗
PubMedDate not supplied

Effects of rare earth compounds on growth and apoptosis of leukemic cell lines.

To explore a new agent for inhibiting leukemic cells, we investigated the effects of rare earth compounds (lanthanum chloride and cerium chloride) on the growth and apoptosis of HL-60 and NB4 cells. The growth of HL-60 and NB4 cells was tested by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) colorimetric assay. The apoptosis was measured by light microscopy, flow cytometry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) method. The effect of LaCl(3) on normal bone marrow hematopoietic progenitor cells was evaluated by colony-forming unit-granulocyte-macrophage (CFU-GM) assay. Under our experimental conditions, MTT assay showed that 48-h treatment with 1, 2, and 3 mM LaCl(3) or 48- and 72-h treatments with 1 mM LaCl(3) could significantly inhibit the growth of HL-60 cells. Treatment with 2 and 4 mM CeCl(3) for 72 h could significantly inhibit the growth of NB4 cells. Apoptosis could be detected on treatment with 2 mM LaCl(3) for 24 h in HL-60 cells by light microscopic morphology examination, flow cytometric analysis, and TUNEL method. Apoptosis could be also detected on treatment with 2 mM CeCl(3) for 72 h in NB4 cells. Treatment with 1 mM LaCl(3) could arrest the transitions from G0/G1 to S phase. The granulocyte-macrophage colony formation of normal bone marrow cells was not significantly inhibited at lower concentrations of LaCl(3) (0.5 to 2 mM). Our results indicate that at certain concentrations, the rare earth compounds may inhibit the growth of leukemic cells, induce them to apoptosis, and have no significant inhibitory effects on normal bone marrow hematopoietic progenitor cells (CFU-GM). The mechanism needs to be further investigated.

Apoptosis↗
PubMed1995

Investigation of cytotoxic effects of 8 norbornane derivatives on 4 human cancer cell lines using the MTT assay.

A group of 8 structurally modified norbornane analogs, selected on basis of water solubility, was tested for its potential cytotoxic effects using a modified MTT assay. Four tumor cell lines, three hematological and a hepatocellular one were used as an experimental model system. Some analogs exhibited an antitumor effect which was shown to be tissue specific. The cytotoxic effect is most likely due to the nature of the side chains rather than to the basic bicyclic norbornane structure of the molecule. These results not only provide a basis for the synthesis of derivatives with effective antitumor activity, but also give some indication of the the molecular target of these compounds.

Antineoplastic Agents↗
PubMed1994

A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro.

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.

Animals↗
PubMed2003

Modulation of the arsenic effects on cytotoxicity, viability, and cell cycle in porcine endothelial cells by selenium.

The differential effects of arsenic compounds and the effect of selenium on arsenic-induced changes in cytotoxicity, viability, and cell cycle of porcine aorta endothelial cells (PAECs) were investigated. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay indicated that arsenic trioxide (As(2)O(3)) and sodium arsenite (NaAsO(2)) showed similar cytotoxicity, whereas sodium arsenate (Na(2)HAsO(4)) did not show cytotoxicity in PAECs. As(2)O(3) and NaAsO(2) at 20 microM decreased PAEC viability, decreased G0/G1 phase, and increased apoptosis. An increased G2/M phase was observed in NaAsO(2)-treated PAECs, whereas an increase in secondary necrosis (late apoptosis) was observed in As(2)O(3)-treated PAECs. As(2)O(3)-induced apoptosis was associated with upregulation of p53 and caspase 3, whereas NaAsO(2)-induced apoptosis was associated with p53 upregulation. Sodium selenite (Na(2)SeO(3)) at 1 nM reduced 20 microM As(2)O(3)-induced cytotoxicity, but not apoptosis, at 24 h. Increased glutathione peroxidase (GPX) activity by Na(2)SeO(3) pretreatment in 20 microM As(2)O(3)-treated PAECs suggests that Na(2)SeO(3) modulates As(2)O(3)-induced cytoxicity by GPX modulation.

Animals↗
PubMed2000

Mercury intolerance in relation to superoxide dismutase, glutathione peroxidase, catalase, and the nitroblue tetrazolium responses.

Through percutaneous provocation with metallic mercury and phenyl mercuric acetate in patients stating the presence of subjective psychosomatic symptoms following dental amalgam treatment, it has been possible to categorize and score two extreme groups of patients, mercury-intolerant and mercury-tolerant patients reacting and not reacting, respectively, to low doses of mercury. The intolerant patients had a high psychosomatic score and the tolerant patients had a low or null score when exposed to low doses of the two mercury compounds. Determination of the scavenger enzymes superoxide dismutase, glutathione peroxidase, and catalase showed no significant differences between the mercury-intolerant and the mercury-tolerant patients and the controls. The activity of superoxide dismutase and the quantitative psychosomatic score elicited by either metallic mercury or phenyl mercuric acetate showed a positive correlation. On the other hand, analyses of the psychosomatic score and the areas under the curves of the nitroblue tetrazolium test response showed negative correlations. The results indicate that the oxidative metabolism and, in particular, superoxide dismutase may be perturbed in mercury-intolerant patients.

Aged↗
PubMed2000

Detection and differentiation of Campylobacter jejuni and Campylobacter coli in broiler chicken samples using a PCR/DNA probe membrane based colorimetric detection assay.

Campylobacter enteritis in humans has been linked to consumption of poultry meat. Surveys show that 30-100% of poultry harbour Campylobacter as normal flora of the digestive tract which indicates a need to identify prevalent organism types in flocks and trace their epidemiology. In this study we describe a Campylobacter genus specific polymerase chain reaction (PCR) assay, amplifying the 16 S-23 S rRNA intergenic spacer region with an internal Campylobacter genus specific DNA probe and species specific probes for Campylobacter jejuni and Campylobacter coli designed for confirmation of the amplified PCR products by Southern blot and colorimetric reverse hybridization assays. The specificity of this assay was established by testing a range of food pathogens. Broiler chicken samples were tested following presumptive positive identification by the Malthus System V analyser (Malthus Instruments, UK). The combined PCR and colorimetric reverse hybridization assay is easy to perform and faster than conventional methods for confirmation and identification of Campylobacter species.

Animals↗
PubMed2005

Synthesis and evaluation of double bond substituted combretastatins.

A series of combretastatins substituted with epoxides, amides and small alkyl groups has been synthesised and evaluated for cytotoxicity and their ability to inhibit the assembly of tubulin. The methyl and ethyl substituted phenols 36, 44 have shown potent antimitotic effects whilst exhibiting reduced cytotoxicity.

Amides↗
PubMed2006

Phase-sensitive polymer-based controlled delivery systems of leuprolide acetate: in vitro release, biocompatibility, and in vivo absorption in rabbits.

Leuprolide acetate (LA) is a synthetic analog of gonadotropin releasing hormone. It is effective in prostate cancer treatment only when its desired concentration in blood is maintained for longer duration. Therefore, the purpose of this study was to investigate the in vitro release, biocompatibility, and in vivo absorption of LA from phase-sensitive polymer delivery systems capable of delivering it at a controlled rate for longer duration. Phase-sensitive formulations were prepared by dissolving dl-polylactic acid (dl-PLA) in a mixture of organic solvents, benzyl benzoate (BB) and benzyl alcohol (BA). LA was incorporated into the polymer solution by homogenization. In vitro release was studied into 15 ml of releasing media contained in a vial which was maintained at 37 degrees C in a reciprocal shaking water bath. The amount of LA in the released samples was analyzed by stability indicating HPLC method. The biocompatibility of polymer formulations was investigated by in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In vivo absorption of LA from delivery systems was studied in rabbits. Blood samples were analyzed for LA and testosterone contents by commercially available immunoassay kits. In vitro release studies showed a greater release of LA from formulations containing a greater proportion of BA (hydrophilic fraction) in the solvent mixture. In vitro biocompatibility study showed significantly (p<0.05) higher cell viability in growth media diluted with polymer extract than the control. In vivo absorption of LA and its effect on testosterone level in rabbits from the delivery system showed a sustained plasma level of LA up to 12 weeks which suppressed the testosterone plasma concentration to castration level beginning from 14th day until 90 days. Thus, phase-sensitive polymer delivery systems of LA were biocompatible and delivered LA at a controlled rate both in vitro and in vivo to keep testosterone plasma concentration at a castration level up to 3 months.

Animals↗
PubMed1980

Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid.

The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, proliferates continuously in suspension culture and consists predominantly (greater than 90%) of promyelocytes. These cells can be induced to differentiate to morphologically and functionally mature granulocytes by incubation with a wide variety of compounds, including butyrate and hypoxanthine and polar planar compounds such as dimethyl sulfoxide and hexamethylene bisacetamide. We have now found that retinoic acid (all-trans-retinoic acid) induces differentiation (as measured morphologically and by the ability to reduce nitroblue tetrazolium) of HL-60 at concentrations as low as 1 nM. Maximal differentiation (approximately 90%) occurs at 1 micro M, a concentration 1/500th to 1/160,000th the concentrations of butyrate (0.5 mM) and dimethyl sulfoxide (160 mM) that promote a similar increase in differentiation. Continuous exposure to retinoic acid is necessary for optimal differentiation, with the percentage of mature cells in the culture directly related to the length of time of exposure to retinoic acid. Retinoic acid and 13-cis-retinoic acid are equally effective in inducing differentiation of HL-60. Retinol (vitamin A), retinal, and retinyl acetate are approximately 1/1000th less potent. This study suggests that retinoids could provide a therapeutic tool in the treatment of acute myeloid leukemia, a disease that has been looked upon as primarily involving a block in myeloid differentiation, and indicates that retinoids, in addition to their well-characterized involvement in epithelial cell differentiation, may also be involved in the differentiation of certain hematopoietic cells.

Cell Differentiation↗