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Structure-staining relationships in histochemistry and biological staining. I. Theoretical background and a general account of correlation of histochemical staining with the chemical structure of the reagents used.

A description is given of the choice, rationale and calculation of several numerical coefficients for describing the chemical structures of staining reagents such as dyestuffs, enzyme substrates, diazonium and tetrazolium salts. Using such coefficients, structure-staining correlations may be made for many staining systems. Examples include the structural correlations of the quantitative affinity measurements of the staining of chromatin with basic dyes, the tetrazolium salts for use as visualizing agents in enzyme histochemistry, and the use of bis-azo compounds as acid or basic dyes, or as fat stains. The value of such structure-staining correlation models for assisting the rational selection and design of new histochemical reagents, and for understanding old reagents and procedures, is discussed.

Chemical Phenomena↗

In vitro cytotoxicity of norditerpenoid alkaloids.

Forty-three norditerpenoid alkaloids isolated from Aconitum, Delphinium and Consolida species have been evaluated for their cytotoxic effects on the tumor cell lines CT26 (murine colon adenocarcinoma), SW480 (human colon adenocarcinoma), HeLa (human cervical adenocarcinoma), SkMel25 (human melanoma) and SkMel28 (human malignant melanoma) with several multidrug resistance mechanisms and the non-tumor cell line CHO (Chinese hamster ovary cells). Neoline (5), 8-O-methylcolumbianine (6), 1,14-diacetylcardiopetaline (9), 18-O-demethylpubescenine (13), 14-deacetylpubescenine (14), pubescenine (15), 14-deacetylajadine (25), lycoctonine (26), browniine (28), delphatine (29), dehydrotakaosamine (34), and ajadelphinine (37) exhibited selective cytotoxicity to cancerous versus non-cancerous cells. Some of these compounds had an irreversible effect on SW480 (5, 15, 25, 26, and 34), HeLa (15, 34, and 37) and SkMel25 (15 and 34) cell lines. In order to gain insights into the mechanism of irreversible cytotoxic action of these compounds we compared the cell viability by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and the acid phosphatase (AP) methods. Our results suggest that the effects of these compounds could be related to the inhibition of ATP production.

Acid Phosphatase↗

The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studied in vitro and with a model system of polyacrylamide films.

The applicability of phenazine methosulfate, 1-methoxyphenazine methosulfate, menadione, and meldola blue as exogenous electron carriers for the cytochemical staining of nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent dehydrogenases has been studied quantitatively with tetranitro BT in vitro and with a model system of polyacrylamide films incorporating either purified glucose-6-phosphate dehydrogenase or intact rat liver parenchymal cells. It was found that every assay in which a tetrazolium salt is used, whether or not an electron carrier is present, has to be carried out in darkness. Menadione did not appear to be useful, because electrons were not found to be transferred directly from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) to this compound. Phenazine methosulfate at higher concentrations and meldola blue at concentrations optimal for carrying electrons to tetrazolium salts yielded a high level of "nothing dehydrogenase" activity in cell-containing films, but no inhibition of enzymatic activity was found. Factors involved in the interference of oxygen with tetrazolium salt reduction are discussed. 1-Methoxyphenazine methosulfate did not stain cellular compounds and caused only a very low nothing dehydrogenase activity. The cytochemical demonstration of dehydrogenase activity was shown to be independent on the concentration of 1-methoxyphenazine methosulfate used (50-1000 microM). It is concluded that 1-methoxyphenazine methosulfate is the exogenous electron carrier of choice.

Acrylic Resins↗

Mechanism of fructosamine assay: evidence against role of superoxide as intermediate in nitroblue tetrazolium reduction.

We studied the chemistry of the fructosamine assay for glycated serum proteins by using the model Amadori compound N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Free lysine was formed at approximately 70% yield during a standard 20-min incubation of fFL with alkaline nitroblue tetrazolium (NBT) at 37 degrees C. Although superoxide dismutase (SOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) decreased the yield of the product, monoformazan dye (MF+), the yield of MF+ was slightly greater under anaerobic than aerobic conditions, excluding a role for superoxide as an intermediate in the reduction of NBT during the fructosamine assay. SOD added to diabetic patients' sera at physiological concentrations also caused a significant (approximately 50%) inhibition of MF+ formation. This inhibition was reduced by addition of nonionic detergents, which contain organic peroxide inhibitors of SOD, to the fructosamine reagent. Overall, these data indicate that the Amadori compound is the direct reductant of NBT in the fructosamine assay and that superoxide is not an intermediate in the reaction. The inhibitory effects of SOD and catalase are most likely the result of oxygen regeneration in the assay mixture.

Catalase↗

Aromatase activity modulation by lindane and bisphenol-A in human placental JEG-3 and transfected kidney E293 cells.

Aromatase is the cytochrome P-450 involved in converting androgens to estrogens. The cytochrome P-450 family plays a central role in the oxidative metabolism of compounds including environmental pollutants. Since lindane and bisphenol-A (BPA) are two well-characterized endocrine disruptors that have been detected in animals and humans, it was important to learn whether they could affect aromatase activity and consequently estrogen biosynthesis. The present study investigates the effects of BPA and lindane on cytotoxicity, aromatase activity and mRNA levels in human placental JEG-3 cells and transfected human embryonal kidney 293 cells. Both cell lines were exposed to increasing concentrations of lindane (25, 50 and 75 microM) and bisphenol-A (25, 50 and 100 microM) over different time periods (10 min-18 h). As a result, none of these concentrations showed cytotoxicity. After short pre-incubation times (10 min-6 h), aromatase activity was enhanced by both compounds. Longer time incubation (18 h), however, produced dose-related inhibition. Lindane and BPA had no significant effects on CYP19 mRNA levels. Therefore, lindane and BPA modulate aromatase activity suggesting an interaction with the cytochrome P-450 aromatase. This study highlights the endocrine-modulating properties of lindane and bisphenol-A.

Adult↗

Volume, conductivity, and scatter changes of activated polymorphonuclear leukocytes: an estimation by Coulter Counter STKS analyzer.

Suspensions of phorbol myristate acetate-activated polymorphonuclear leukocytes were analyzed with a Coulter Counter STKS hematological analyzer. Phorbol myristate acetate activation induced an increase in polymorphonuclear leukocyte volume and conductivity, while scatter was unchanged. Phorbol myristate acetate-activated neutrophils in a suspension containing nitroblue tetrazolium showed increased scatter. The rise in scatter was phorbol myristate acetate dose dependent, completely inhibited by diphenylene iodonium and partially by dimethyl sulfoxide, two inhibitors of NADPH oxidase. Zymosan-activated polymorphonuclear leukocytes were notably larger with a characteristic position on discriminant function 1 display (volume versus scatter) of the analyzer. Volume and conductivity changes were seemingly inexplicable features of phorbol myristate acetate activation. The rise in scatter was produced by cytoplasmic precipitation of reduced nitroblue tetrazolium and thus by O2-generation in phorbol myristate acetate-activated neutrophils. Zymosan phagocytosis was responsible for the notable rise in polymorphonuclear leukocyte volume. The analysis of activated polymorphonuclear leukocytes by Coulter Counter STKS may provide useful information on their activation and a pragmatic approach for studying function.

Biphenyl Compounds↗

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely used screening method to measure cell viability and proliferation. When testing the effects of kaempferol on breast cancer cell number (crystal violet staining) and viability (MTT tetrazolium assay) conflicting results were obtained. Cell number decreased but MTT formazan formation increased, suggesting a direct interaction of kaempferol with the MTT tetrazolium reduction. Direct reductive potential was observed in a cell-free system for the presumptive phytoestrogens kaempferol and resveratrol, and extracts of Hypericum perforatum L. and Cimicifuga racemosa L. All agents led to instantaneous dark blue formazan formation in the absence of cells. Additionally, antioxidants such as ascorbic acid, vitamin E and N-acetylcysteine interfered with the MTT tetrazolium assay. When MCF7 and HS578 cells treated with kaempferol were washed before addition of MTT tetrazolium, the direct reduction of dye was reduced significantly. These results indicate that the MTT tetrazolium assay may lead to false positive results when testing natural compounds with intrinsic reductive potential.

Acetylcysteine↗

Induction of differentiation of human myeloid leukemia HL-60 cells by novel pyrimidine nucleoside analogs.

New pyrimidine nucleoside analogs (18 compounds) were synthesized and their growth-inhibiting and differentiation-inducing activities on human myeloid leukemia HL-60 cells were examined. Some of the analogs were found to induce nitroblue tetrazolium (NBT) reducing activity in the HL-60 cells. The inducing activities of these compounds were compared at their concentrations for 50% inhibition of cell growth. TI-79 (3-benzyl-5-methyl-3-(beta-D-ribofuranosyl)pyrido[2,3-d]pyrimidine- 2,4(1H,3H)-dione) was a very effective inducer of NBT-reduction and of differentiation of the cells into mature granulocytes. The induction of NBT-reducing activity by TI-79 was inhibited by high concentrations of the natural nucleoside, adenosine. Other differentiation inducers, such as retinoic acid, 1 alpha,25-dihydroxyvitamin D-3 and staurosporin markedly enhanced the induction of differentiation of HL-60 cells by TI-79. Nucleoside analogs such as TI-79 should be useful for differentiation therapy of some types of myelogenous leukemia.

Adenosine↗

Factors influencing the entry of dye into neutrophil leucocytes in the Nitroblue Tetrazolium test.

1. The Nitroblue Tetrazolium (NBT) test is a measure of the phagocytosis of particulate complexes of NBT and heparin and/or fibrinogen by neutrophil leucocytes. 2. Humoral factors in the plasma of ill patients stimulate neutrophil leucocytes of normal subjects to ingest these complexes. 3. The acute phase protein, orosomucoid and endotoxin stimulate reduction of NBT in vitro and could be responsible for some positive NBT tests in vivo. 4. The effect of factors promoting phagocytosis may be masked by an inability of neutrophil leucocytes to respond to stimulation. 5. This defective neutrophil function could result from the replacement of circulating neutrophil leucocytes by less mature, less actively phagocytic cells or by the presence of circulating immune complexes.

ABO Blood-Group System↗

Comparison of the toxicities of cisplatin and a new cisplatin-procaine complex to rat renal cortical slices.

1. Procaine has previously been shown to diminish the nephrotoxicity of cisplatin and the nephrotoxic effects of cisplatin and a new cisplatin complex (cis-diamminechloro-[2-(diethylamino) ethyl-4-aminobenzoate, N4]-chlorideplatinum (II) monohydrochloride monohydrate; DPR), that contains procaine hydrochloride were compared with rat renal cortical slices. 2. Cisplatin at 1 mM caused toxicity to the slices, as shown by an increase in the leakage of aspartate aminotransferase and lactate dehydrogenase from the slices into the incubation medium and a decrease in the reduction of a tetrazolium dye (MTT assay). Addition of procaine (1 mM) protected against cisplatin-induced toxicity. DPR either at 1 mM or at 4 mM had no effect either on the enzyme leakage or MTT reduction by the renal slices, but DPR at 10 mM produced a similar magnitude of enzyme leakage to cisplatin (1 mM). 3. DPR lowered the concentration of ATP and glutathione (GSH) in the slices but was less potent than cisplatin. Thiobarbituric acid reactive substances, indicators of lipid peroxidation, released into the medium were increased by the highest concentration of DPR (10 mM), which suggests that DPR has the potential to cause oxidative stress. 4. The results suggest that DPR was far less toxic than either cisplatin alone or a mixture of cisplatin and procaine.

Adenosine Triphosphate↗

Measurement of superoxide release from single pulmonary alveolar macrophages.

An electrooptical method was developed to quantify superoxide (O2-) release from single rat pulmonary alveolar macrophages (PAM) during adherence to the bottom of a culture dish. This was done by measuring the reduction of nitro blue tetrazolium (NBT) to a diformazan precipitate at 550 nm from videorecorded images of individual cells. Temporal changes in cell optical density, which are proportional to the mass of diformazan produced, were calculated from videophotometric measurements of the change in light intensity over individual cells. Total diformazan produced increased 78 and 126% with an increase in NBT from 0.5 to 1.0 and 2.0 mg/ml, respectively. Total diformazan produced and maximum rate of production among individual PAM varied two- to threefold providing strong evidence for heterogeneity in O2- production. Specific inhibition of O2- production by superoxide dismutase, iodoacetate, and chlorpromazine significantly reduced the total diformazan produced and maximum rate of diformazan production. Hydrogen peroxide was not involved in NBT reduction, since catalase alone did not significantly change diformazan production. This novel method to quantify O2- release from single PAM should be valuable in analyzing heterogeneity and single cell kinetics of O2- production, in assessing the effects of exposure of cells to particulates on O2- release, and in relating release to electrophysiological measurements.

Animals↗

Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR).

Visualization of bacteriophage plaques may be enhanced by addition of ferric ammonium citrate and sodium thiosulfate (FACST) or 2,3,5-triphenyltetrazolium chloride (tetrazolium red, TZR) to the soft agar layer of a traditional bacteriophage plaque assay. Background color from these reagents improved contrast between clear plaques and turbid host lawns in trypticase soy agar (TSA) plates. Enhancement by FACST is based on reaction with hydrogen sulfide gas (H2S) produced by some strains of bacteria and was tested here using H2S+ and H2S- strains of Salmonella enterica subsp. enterica with a bacteriophage (Podoviridae) isolated from swine lagoon effluent. Only the H2S+ strain produced dark brown-black color in FACST-amended agar. Both strains showed bright pinkish-red color in TZR-amended agar. Color intensity for both reagents decreased with decreasing concentrations of the reagents. Contrast in FACST-amended plates appeared greater than that with TZR, but diminished after 12 h, while contrast in TZR-amended plates remained constant. At the concentrations tested, neither reagent affected plaque counts in the H2S+ strain. The FACST should be useful in bacteriophage plaque assays with H2S+ strains of Salmonella and other H2S+ bacteria.

Bacteriological Techniques↗

Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT).

The oxidation of arachidonic acid by ferrous sulfate provides a useful model to study the role of iron in lipid oxidation reactions. We have employed nitroblue tetrazolium (NBT) in the present investigation to evaluate the mechanism of this reaction. In the presence of arachidonic acid, Fe +++, and O2, the yellow dye NBT was rapidly reduced to the blue form, NBTH2. The molar ratio of arachidonic acid to Fe++ in this rapid reaction was 1:1, showing an interaction of one fatty acid molecule per iron molecule. Approximately one molecule of NBT was reduced per four molecules of arachidonic acid and Fe++. Reduction of NBT was accompanied by oxidation of Fe++ to Fe+++, suggesting the transfer of four electrons from the Fe++ to NBT to reduce the dye. Arachidonic acid was found to be unchanged when extracted at the end of the reaction, indicating formation of a complex that could dissociate leaving intact arachidonic acid. Evidence for the presence of such a complex which slowly dissociates during the reaction was obtained after longer incubations with small amounts of arachidonic acid. NBT reduction was not inhibited by agents which hydrolyze superoxide, by catalase or by agents which trap hydroxy radicals. We, therefore, propose a model in which NBT traps a radical generated on the arachidonic acid molecule. The proposed model suggests mechanisms for other fatty acid oxidation reactions such as prostaglandin and hydroperoxy fatty acid synthesis.

Arachidonic Acids↗