Kinetic colorimetric measurement of serum lactate dehydrogenase activity.
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Azo Compounds↗
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Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells.
BACKGROUND: Certain antimitotic drugs have antitumor activities that apparently result from interactions with nontubulin components involved in cell growth and/or apoptotic cell death. Indanocine is a synthetic indanone that has been identified by the National Cancer Institute's Developmental Therapeutics Program as having antiproliferative activity. In this study, we characterized the activity of this new antimitotic drug toward malignant cells. METHODS: We tested antiproliferative activity with an MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, mitochondrial damage and cell cycle perturbations with flow cytometry, caspase-3 activation with fluorometry, alterations of the cytoskeletal components with immunofluorescence, and antimicrotubule activity with a tubulin polymerization assay. RESULTS/CONCLUSIONS: Indanocine is a cytostatic and cytotoxic indanone that blocks tubulin polymerization but, unlike other antimitotic agents, induces apoptotic cell death in stationary-phase multidrug-resistant cancer cells at concentrations that do not impair the viability of normal nonproliferating cells. Of the seven multidrug-resistant cell lines tested, three (i.e., MCF-7/ADR, MES-SA/DX5, and HL-60/ADR) were more sensitive to growth inhibition by indanocine than were their corresponding parental cells. Confluent multidrug-resistant cells (MCF-7/ADR), but not drug-sensitive cancer cells (MCF-7) or normal peripheral blood lymphocytes, underwent apoptotic cell death 8-24 hours after exposure to indanocine, as measured by sequential changes in mitochondrial membrane potential, caspase activity, and DNA fragmentation. Indanocine interacts with tubulin at the colchicine-binding site, potently inhibits tubulin polymerization in vitro, and disrupts the mitotic apparatus in dividing cells. IMPLICATIONS: The sensitivity of stationary multidrug-resistant cancer cells to indanocine suggests that indanocine and related indanones be considered as lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.
Antineoplastic Agents↗
Sulfated glycosaminoglycans and dyes attenuate the neurotoxic effects of beta-amyloid in rat PC12 cells.
Glycosaminoglycan (GAG)-containing proteoglycans are associated with the neuritic plaques and cerebrovascular beta-amyloid deposits of Alzheimer's disease as well as with the amyloid deposits of prion and other disorders. GAGs and other sulfate-containing compounds have previously been shown to bind beta-amyloid peptide in vitro, suggesting possible effects of beta-amyloid deposition and/or toxicity in vivo. Using reduction of the redox dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure beta-amyloid neurotoxicity in rat pheochromocytoma PC12 cells, several polysulfated GAGs and synthetic sulfate-containing compounds were found to attenuate the neurotoxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.
Amyloid beta-Peptides↗
3,4,5,3',5'-Pentabromo-2-(2'-hydroxybenzoyl)pyrrole: a potential lead compound as anti-Gram-positive and anti-biofilm agent.
The activity against Gram-positive bacteria of 3,4,5,3',5'-pentabromo-2-(2'-hydroxybenzoyl)pyrrole I, a synthetic anti-bacterial compound related to pyrrolomycins, was tested in vitro using seven reference bacterial strains and Staphylococcus epidermidis and Staphylococcus aureus preformed biofilms. Compound I was active against all strains tested, with minimum inhibitory concentration (MIC) values ranging from 0.002 to 0.097 mg/l and minimum bactericidal concentrations (MBCs) from 0.37 to 12.5 mg/l. Compound I was also active at low concentrations against preformed S. epidermidis and S. aureus biofilms.
Anti-Bacterial Agents↗
Inhibition of beta-amyloid toxicity by short peptides containing N-methyl amino acids.
Single N-methyl amino acid-containing peptides related to the central hydrophobic region beta16-20 (Lys-Leu-Val-Phe-Phe) of the beta-amyloid protein are able to reduce the cytotoxicity of natural beta1-42 in PC12 cell cultures. N-methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t-test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.
Alzheimer Disease↗
Tomato paste fraction inhibiting the formation of advanced glycation end-products.
A water-soluble and low-molecular-weight fraction (SB) was obtained from tomato paste. The effects of SB on the formation of advanced glycation end-products (AGE) in protein glycation were studied by the methods of specific fluorescence, ELISA and a Western blot analysis, using the anti-AGE antibody after incubating protein with sugar. The results suggest that SB had strong inhibitory activity, in comparison with aminoguanidine as a positive control, and that the inhibitory mechanism of SB differed from that of aminoguanidine to involve trapping of reactive dicarbonyl intermediates in the early stage of glycation. SB contained an antioxidant, rutin, which showed potent inhibitory activity. The results also suggest that rutin chiefly contributed to inhibiting the formation of AGE, and that other compounds in SB may also have been related to the activity.
Arginine↗
[Activated sludge activity: an easy test for inspection of biological purification plants].
The results of the enzymatic study of microbial dehydrogenases with triphenyl tetrazolium chloride (TTC) present the possibility of a new practical method which can be used in activated-sludge plants. The procedure has been modified, with regard to simplicity and low cost of operation.
Oxidoreductases↗
Cytotoxic activity of Justicia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in a cell cycle dependent fashion.
Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation.
Acanthaceae↗
Pig Leydig cell culture: a useful in vitro test for evaluating the testicular toxicity of compounds.
In vivo studies have shown that the testis is a target organ for drugs and chemicals. In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, we have developed a miniaturized primary culture of immature pig Leydig cells. Five well-known drugs with differing mechanisms of toxicity on testicular functions were tested to validate the model. Testosterone and progesterone secretion were measured to evaluate testicular function. Cell viability was assessed quantitatively using a colorimetric assay based on the reduction of a tetrazolium salt (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) which stains viable cells only, thus allowing discrimination between specific inhibitors of Leydig cell function and nonspecific cytotoxic drugs. Ketoconazole and aminoglutethimide inhibited both testosterone and progesterone secretion, but without modifying cell viability. Spironolactone specifically blocked testosterone secretion and increased progesterone concentration without inducing cell mortality. Cycloheximide altered testicular steroid secretion by another mechanism of action. Chlorpromazine, which interferes with the secretion of gonadotropins in vivo, produced a significant inhibition of progesterone and testosterone secretion as a result of the cytotoxic effects of the drug. In conclusion, this in vitro test enables one to discriminate accurately between specific and nonspecific inhibitors of steroidogenesis and could reduce the number of false positives when screening for potential testicular toxins.
Aminoglutethimide↗
Iron autoxidation in Mops and Hepes buffers.
Iron autoxidation in Mops and Hepes buffers is characterized by a lag phase that becomes shorter with increasing FeCl2 concentration and pH. During iron oxidation in these buffers a yellow colour develops in the solution. When the reaction is conducted in the presence of nitro blue tetrazolium (NBT), blue formazan is formed. Of the many OH scavengers tested, mannitol and sorbitol are most effective in inhibiting Fe2+ oxidation, yellow colour development and NBT reduction. Some inhibition was also noted with catalase. The iron product of the oxidative reaction differs from Fe3+ in its absorption spectrum and its low reactivity with thiocyanate. Similar results are obtained when iron autoxidation is studied in unbuffered solutions brought to alkaline pH with NaOH. In phosphate buffer, no lag phase is evident and the absorption spectrum of the final solution is identical to that of Fe3+ in this buffer. The iron product reacts immediately with thiocyanate. When iron oxidation is conducted in the presence of NBT the formation of formazan is almost undetectable. Of the many compounds tested only catalase inhibits iron autoxidation in this buffer. The sequence of reactions leading to iron autoxidation in Good-type buffers thus resembles that occurring in unbuffered solutions brought to alkaline pH with NaOH and greatly differs from that occurring in phosphate buffer. These results are in agreement with the observation that these buffers have very low affinity for iron. The data presented define experimental conditions where Fe2+ is substantially stable for a considerable length of time in Mops buffer.
Buffers↗
Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5.
The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor). In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously. Kinetic investigations of the redox couple FdRmor/Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fdmor in this reaction, compared with the NADH-dependent reduction of cytochrome c. The quite low Km (5.3 +/- 0.3 nm) of FdRmor for Fdmor, measured with NBT as the electron acceptor, indicated high specificity. The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins. Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form, which has previously not been possible for wild-type P450mor. The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under any conditions. Pyridine, metyrapone and different azole compounds generated type II binding spectra and the Kd values determined for these substances suggested that P450mor might have a preference for more bulky and/or hydrophobic molecules. The purified recombinant proteins FdRmor, Fdmor and P450mor were used to reconstitute the homologous P450-containing mono-oxygenase, which was shown to convert morpholine.
Catalysis↗
Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.
Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.
3T3 Cells↗
Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60.
Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.
Animals↗
Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells.
Lycopene, the major tomato carotenoid, has been found to inhibit proliferation of several types of cancer cells, including those of breast, lung, and endometrium. By extending the work to the HL-60 promyelocytic leukemia cell line, we aimed to evaluate some mechanistic aspects of this effect. Particularly, the possibility was examined that the antiproliferative action of the carotenoid is associated with induction of cell differentiation. Lycopene treatment resulted in a concentration-dependent reduction in HL-60 cell growth as measured by [3H]thymidine incorporation and cell counting. This effect was accompanied by inhibition of cell cycle progression in the G0/G1 phase as measured by flow cytometry. Lycopene alone induced cell differentiation as measured by phorbol ester-dependent reduction of nitro blue tetrazolium and expression of the cell surface antigen CD14. Results of several recent intervention studies with beta-carotene, which have revealed no beneficial effects of this carotenoid, suggest that a single dietary component cannot explain the anticancer effect of diets rich in vegetables and fruits. Thus another goal of our study was to examine whether lycopene has the ability to synergize with other natural anticancer compounds, such as 1,25-dihydroxyvitamin D3, which when used alone are therapeutically active only at high and toxic concentrations. The combination of low concentrations of lycopene with 1,25-dihydroxyvitamin D3 exhibited a synergistic effect on cell proliferation and differentiation and an additive effect on cell cycle progression. Such synergistic antiproliferative and differentiating effects of lycopene and other compounds found in the diet and in plasma may suggest the inclusion of the carotenoid in the diet as a cancer-preventive measure.
Anticarcinogenic Agents↗
The chemical constitution and biocompatibility of accelerated Portland cement for endodontic use.
AIM: To evaluate the biocompatibility of mineral trioxide aggregate and accelerated Portland cement and their eluants by assessing cell metabolic function and proliferation. METHODOLOGY: The chemical constitution of grey and white Portland cement, grey and white mineral trioxide aggregate (MTA) and accelerated Portland cement produced by excluding gypsum from the manufacturing process (Aalborg White) was determined using both energy dispersive analysis with X-ray and X-ray diffraction analysis. Biocompatibility of the materials was assessed using a direct test method where cell proliferation was measured quantitatively using Alamar Blue dye and an indirect test method where cells were grown on material elutions and cell proliferation was assessed using methyltetrazolium assay as recommended by the International standard guidelines, ISO 10993-Part 5 for in vitro testing. RESULTS: The chemical constitution of all the materials tested was similar. Indirect studies of the eluants showed an increase in cell activity after 24 h compared with the control in culture medium (P<0.05). Direct cell contact with the cements resulted in a fall in cell viability for all time points studied (P<0.001). CONCLUSIONS: Biocompatibility testing of the cement eluants showed the presence of no toxic leachables from the grey or white MTA, and that the addition of bismuth oxide to the accelerated Portland cement did not interfere with biocompatibility. The new accelerated Portland cement showed similar results. Cell growth was poor when seeded in direct contact with the test cements. However, the elution made up of calcium hydroxide produced during the hydration reaction was shown to induce cell proliferation.
Aluminum Compounds↗
Synthesis and cytotoxic activity of 1-alkoxy- and 1-amino-2-hydroxy-1,2-dihydroacronycine derivatives.
Sixteen new derivatives of the natural alkaloid acronycine, bearing 1-alkoxy or 1-amino and 2-hydroxy groups, were synthesized in order to clarify the role of the C-1 substitution. Studies on the cytotoxic activity of compounds 4-19 were carried out in vitro on L-1210 cells. Structure-activity relationships are discussed.
Acronine↗
[Partially hydrogenated aryl-1,2/1,4-anthraquinone derivatives, 5-lipoxygenase inhibitors with arotinoid structure].
The combination of 5-LOX inhibition and retinoid activity in one molecule could be an interesting pharmacological tool to influence psoriasis. Thus we synthesized compounds with arotinoid structure by anellation of the 5-LOX inhibitors 1 and 2 with 1,1,4,4-tetramethylcyclohexane. A key step was the CuCl-MeCN-O2 oxidation of tetrahydroanthracenol 13 to the corresponding 1,2-anthraquinone 14 which could be converted to the analogous 2-hydroxy-1,4-anthraquinone 19 by Thiele-Winter reaction followed by oxidation. The halogenated quinones 9 and 21 were arylated with 2,6-di-tertbutylphenol and demethylated or hydrolyzed to the target compounds 3 and 4 which were tested in comparison with the non-anellated 5-LOX inhibitors 1 and 2 for LOX inhibition in activated human granulocytes and for antioxidative activity by the method of Popov with the chemiluminometer Photochem. The results are discussed in relation to the corresponding logP values. The 1,2-quinones 1 and 3 are more potent 5-LOX inhibitors than their 1,4-analogues 2 and 4, the tetrahydroanthraquinon derivatives 3 and 4 are less potent than the naphthoquinones 1 and 2. All compounds are devoid of any activity in cell differentiation as compared to retinoic acid as indicated by the NBT test with HL-60 leukemia cells.
Anthraquinones↗
Glucose-6-phosphate dehydrogenase: the key to sex-related xenobiotic toxicity in hepatocytes of European flounder (Platichthys flesus L.)?
The role of glucose-6-phosphate dehydrogenase (G6PDH) in oxidative stress responses was investigated in isolated intact living hepatocytes of immature female and male European flounder (Platichthys flesus L.) because it is the major provider of NADPH needed as reducing power for various detoxification pathways. Hepatocytes were exposed to sublethal concentrations of effective prooxidants such as 100 microM hydrogen peroxide (H(2)O(2)), 100, 200 and 400 microM benzo[a]pyrene (B[a]p) and 50 microM nitrofurantoin (NF) during culture. Since there is evidence that 17-beta-estradiol inhibits certain pathways of xenobiotic biotransformation, we tested also the effects of different concentrations of 17-beta-estradiol (0.2, 1 and 2 microM) alone and 1 microM in combination with 100 microM B[a]p on G6PDH activity. After short-term (1 day) and long-term (9 days) exposure, G6PDH activity was quantified in intact living hepatocytes by a tetrazolium salt method using tetranitroblue tetrazolium salt (TNBT). Hepatocytes obtained from male fish generally showed higher G6PDH activity than those of females. We observed significant inhibition of G6PDH activity by all oxidative stressors and 17-beta-estradiol in both sexes of fish independently of culture conditions, but inhibition was stronger in cells of females than in cells of males. A cumulative effect of the steroid and B[a]p was not found. Our results indicate a sex-dependent inhibitory effect of all stressors and 17-beta-estradiol on G6PDH activity in flounder hepatocytes independent of prooxidant activity of the specific compound. Consequently, NADPH supply for xenobiotic detoxification and other cellular antioxidative defence mechanisms may be different in livers of female and male flounder. The strongly decreased supply of NADPH in hepatocytes of females may explain the reduced and/or delayed NADPH-dependent activity of xenobiotic biotransformation systems such as cytochrome P450 (CYP450) and a lower capacity of non-enzymatic defence systems such as reduced glutathione that is particularly observed in female flounder. Moreover, the strong inhibition of G6PDH in livers of female flounder may explain higher susceptibility for xenobiotic toxicity and, therefore, potentially a higher risk to develop liver cancer.
Animals↗