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Formazan cells were less numerous in the peripheral blood of vervet monkeys (Cercopithecus aethiops) receiving a "O" protein-low calorie diet than in animals fed a full or a protein but not calorie deficient diet.
WHI-07, a novel bromo-methoxy-substituted aryl phosphate derivative of zidovudine (ZDV), is a potent dual-function contraceptive agent. Although the bromo-methoxy functional groups in the thymine ring of its ZDV are very important for its sperm-immobilizing activity (SIA), the importance of the esterification of the phosphate group with an amino acid side chain and the identity of the para substituent in the aryl moiety remain unclear. In the present study, we have synthesized 23 new analogues of WHI-07 by replacing the alanine (Ala) side chain with different amino acids containing nonpolar side chains, namely tryptophan (Trp), proline (Pro), phenylalanine (Phe), leucine (Leu), methionine (Met), valine (Val), or glycine (Gly). The para substituents on the aryl moiety included bromo, chloro, fluoro, nitro, or methoxy groups. The SIA of each of the 23 WHI-07 analogues was evaluated by computer-assisted sperm analysis. The potential cytotoxicity of these compounds against normal human ectocervical and endocervical epithelial cells was evaluated using MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assays. The replacement of the Ala side chain of WHI-07 with Val, Leu, or Phe led to a complete loss of SIA (EC(50) values > 500 microM), whereas replacement with Trp reduced the SIA by 4-fold. The presence of para substituents on the phenyl moiety led to significant alterations in SIA. The anti-human immunodeficiency virus (HIV) activity of Trp-containing WHI-07 analogues was also diminished. Our finding highlights the necessity of Ala side chain and the presence of electron-withdrawing para-bromo substituent on the phenyl moiety in addition to bromo-methoxy functionalization groups on the thymine ring in order for the phosphoramidate derivatives of ZDV to be effective dual-function spermicidal agents. Unlike the detergent-type microbicide, nonoxynol-9, which was cytotoxic to normal human ectocervical and endocervical epithelial cells (IC(50) values of 22 microM and 16 microM, respectively) at spermicidal concentrations (EC(50) = 81 microM), WHI-07 and its active analogues were selectively spermicidal without cytotoxicity against female genital tract epithelial cells. WHI-07 and its Trp analogues hold particular clinical promise for the development of novel, nondetergent-type prophylactic contraceptives for the prevention of heterosexual HIV/acquired immunodeficiency syndrome transmission.
RATIONALE AND OBJECTIVES: This study compared the areas demarcated by a T1-enhancing agent, Gd-DTPA-BMA, and a magnetic susceptibility agent, Dy-DTPA-BMA, with 201thallium autoradiography (indicator of perfusion) and postmortem histochemical staining with triphenyltetrazolium chloride (TTC)(indicator of infarction). METHODS: Thirteen rats were subjected to coronary artery occlusion for 3 to 4 hours before acquisition of four sets of electrocardiogram-gated spin-echo magnetic resonance (MR) images: T1-weighted images before and after 0.2 mmol/kg Gd-DTPA-BMA; and T2-weighted images before and after 0.3 mmol/kg Dy-DTPA-BMA. After MR imaging, intravenous 201thallium delineated the area of decreased myocardial perfusion. At autopsy, TTC staining delineated the area of myocardial infarction. RESULTS: A myocardial region in the distribution of the occluded artery was delinated as a hyperintense area ("hot-spot") by Dy-DTPA-BMA and as a hypointense area ("cold-spot") by Gd-DTPA-BMA. The hyperintense area demarcated by Dy-DTPA-BMA (51 +/- 3% of the area of the midequitorial slice of the left ventricle) showed a closer relationship to the area of decreased myocardial perfusion (jeopardized area) (46 +/- 3%), determined by 201thallium autoradiography, than the area of myocardial infarction (36 +/- 4%), determined by histochemical staining. However, the hypointense area demarcated by Gd-DTPA-BMA (29 +/- 2%) did not relate as closely to the area of decreased myocardial perfusion (slope = 0.54) or the area of myocardial infarction (r = 0.46). CONCLUSIONS: The abnormal myocardial area delineated by the magnetic susceptibility agent showed a closer relationship to the area of deficient myocardial perfusion (jeopardy area) after coronary occlusion than that defined by T1-enhancing contrast media.
Phorbol dibutyrate induced a nitroblue tetrazolium-reducing reaction in differentiated HL-60 cells, which was inhibited by protein kinase inhibitors such as staurosporine and H-7. ID50 of staurosporine and H-7 were 1.4 ng/ml and 0.19 mM, respectively. When tautomycin, an inhibitor of protein phosphatases, was added with the kinase inhibitors, the nitroblue tetrazolium-reducing reaction again appeared. ID50 of staurosporine was 510 ng/ml in the presence of tautomycin. Tautomycin itself weakly induced the reaction, which was inhibited by kinase inhibitors. Such a competitive effect between tautomycin and staurosporine was not observed in a cell-free system of protein kinase C. Okadaic acid had the same effect as tautomycin. The similar results were obtained when respiratory burst was quantitated by measuring H2O2 produced by canine peripheral neutrophils. The mechanism of competitive effect of tautomycin and staurosporine on respiratory burst is discussed.
During the course of measuring superoxide dismutase (SOD) activity in rat breast tissue, interferences in the nitroblue tetrazolium (NBT) and cytochrome c assay systems were noted. These interferences inhibit accurate measurement of SOD activity in breast tissues, necessitating the development of a new NBT-based assay that includes compounds capable of inhibiting tissue specific interferences. The most effective compounds were metal chelators that were also electron transport chain inhibitors. Bathocuproine sulfonate (BCS) was the most effective of these compounds. The inclusion of BCS in the NBT assay system was shown to make the accurate measurement of SOD activity in tissues with interferences possible.
The heats of formation of 1H-imidazole, 1H-1,2,4-trizazole, 1H-tetrazole, CH3NO2, CH3N3, CH3NH2, CH2CHNO2, HClO4, and phenol, as well as cations and anions derived from some of the molecules have been calculated using ab initio molecular orbital theory. These molecules are important as models for compounds used for energetic materials synthesis. The predicted heats of formation of the heterocycle-based compounds are in excellent agreement with available experimental values and those derived from proton affinities and deprotonation enthalpies to <1 kcal/mol. The predicted value for the tetrazolium cation differs substantially from the experimental value, likely due to uncertainty in the measurement. The heats of formation of the nitro and amino molecules, as well as phenol/phenolate, also are in good agreement with the experimental values (<1.5 kcal/mol). The heat of formation of CH3N3 is predicted to be 72.8 kcal/mol at 298 K with an estimated error bar of +/-1 kcal/mol on the basis of the agreement between the calculated and experimental values for DeltaH(f)(HN3). The heat of formation at 298 K of HClO4 is -0.4 kcal/mol, in very good agreement with the experimental value, as well as a W2 literature study. An extrapolation of the CCSD(T)/aug-cc-pV(Q,5) energies was required to obtain this agreement. This result suggests that very large basis sets (> or =aug-cc-pV5Z) may be needed to fully recover the valence correlation energy contribution in compounds containing elements with high formal oxidation states at the central atom. In addition tight d functions are needed for the geometry predictions. Douglas-Kroll-Hess (DKH) scalar relativistic corrections for HClO4 and ClO4- at the MP2 level with correlation-consistent DKH basis sets were predicted to be large, likely due to the high formal oxidation state at the Cl.
Tetrazolium-dye-linked alcohol dehydrogenase (TD-ADH) of Amycolatopsis methanolica could be resolved into three protein components, which have been purified. Each of the components has the ability to reconstitute TD-ADH activity when combined with the other two. Component 1 is identical to the previously characterized methanol:N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MNO), a decameric protein with 50-kDa subunits, each carrying a tightly bound NADPH. Component 2 is a high molecular mass (> 640 kDa) protein with subunits of 44 kDa and 72 kDa, and which possesses a low tetrazolium-dye-linked NADH dehydrogenase activity. The protein contains a yellow chromophore of unknown identity. Component 3 is a low molecular mass (15 kDa) protein containing a 5'-deazaflavin and at least one other low-molecular-mass compound with properties similar, but not identical, to those of nicotinamide coenzymes. The results suggest that alcohol oxidation by the TD-ADH complex is carried out by component 1 (MNO), after which transfer of the reducing equivalents (mediated by component 3) occurs to component 2, which (in vitro) is linked to the tetrazolium dye. Fractionation of A. methanolica extracts showed that most of the 5'-deazaflavin was present in component 3. Other gram-positive bacteria having a TD-ADH complex also produced 5'-deazaflavin. It is concluded that oxidation of primary aliphatic alcohols by A. methanolica, and probably also by other gram-positive bacteria containing MNO or TD-ADH, proceeds via TD-ADH. The likeliness of 5'-deazaflavin participation in this process is discussed.
This study uses the well known sensitive and reproducible human peripheral blood lymphocyte (PBL) assay as a model for the possible discrimination between cytotoxic and/or immunomodulative properties of 5 reference compounds: cadmium chloride (CdCl2): didodecyl-dimethyl ammonium bromide (DDAB); cyclosporin (CsA); dexamethasone (DEXA); and theophylline. Two colorimetric cytotoxicity assays were employed, the thiazolyl blue tetrazolium bromide and neutral red, and the H-thymidine DNA incorporation assay on phytohemagglutinin stimulated or non-stimulated PBL was used, as a parameter of cell mediated immunity. This study demonstrated the importance of performing colorimetric or other cytotoxicity assays before any investigation of mechanisms of action of different substances on immunomodulation and relative human risk assessment. This study showed that CdCl2 and DDAB have a cytotoxic rather than immunomodulative effect on PBL and that CsA, DEXA and to a lesser extent theophylline have immunomodulative rather than cytotoxic effects on PBL within the range of the concentrations studied.
A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., 'nothing dehydrogenase' activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and 'nothing dehydrogenase') when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.
Myocardial tissue is routinely exposed to the vital stain triphenyl tetrazolium chloride (TTC) to delineate infarction in conjunction with myocardial isotope research. However, it is unknown whether TTC has a direct effect on tracer deposition. We evaluated this possibility in rabbit hearts injected with either teboroxime, sestamibi or 201Tl. The hearts were excised and treated as follows: (1) TTC or normal saline was perfused through the heart and the residual activity monitored; (2) hearts were sliced into 0.5-cm thick sections, counted and incubated in either TTC or normal saline for 10 min then recounted; and (3) the circumflex artery was ligated postmortem and TTC perfused. Autoradiographic images were produced from 30-microns slices to depict any disparity in activity concentration from the selective perfusion of TTC. Both perfusion and incubation by TTC resulted in a significant activity loss of both 201Tl and sestamibi, but not teboroxime, compared to normal saline. An independent octanol extraction experiment measured the change in the partition coefficient of labeled teboroxime and sestamibi induced by the addition of TTC. TTC was shown to liberate the radiolabel from sestamibi, but not from teboroxime. We conclude that histochemical staining techniques involving TTC can alter the distribution of radiolabeled pharmaceuticals. As a result, experiments using TTC with 201Tl and sestamibi should be interpreted cautiously.
A novel colorimetric microbial bioassay for toxicity has been developed; it shows particular sensitivity to trichothecene mycotoxins. The assay uses inhibition of expression of beta-galactosidase activity within the yeast Kluyveromyces marxianus as a sensitive toxicity indicator, cultures remaining yellow, rather than turning deep green-blue, in the presence of X-gal, a chromogenic substrate. The assay is conducted in standard microtitre plates, permitting small volumes (160 microl) and many replicates, and can be scored either automatically by a plate-reader, or by eye. Factors likely to affect the efficacy of the bioassay, including carbon source, solvents, inoculum cell density, and the use of membrane-modulating agents (MMAs), were assessed. Polymyxin B nonapeptide was the most effective toxicity-enhancing MMA tested, enabling the trichothecene mycotoxin, verrucarin A, to be detected at a concentration of about 1 ng/ml. The assay's reproducibility was examined using polymyxin B sulfate, a cheaper MMA, and another trichothecene mycotoxin, T2 toxin: reproducibility and sensitivity were better for the beta-galactosidase X-gal endpoint than for an alternative chromogenic toxicity indicator, the respiratory substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).
A rapid colorimetric assay for viability of E. histolytica trophozoites using the tetrazolium salt MTT and phenazine methosulfate (PMS) was developed. The MTT is normally reduced by dehydrogenases of viable cells and transformed to formazan, a colored compound; the reaction is catalyzed by PMS. Trophozoites of E. histolytica were incubated in the presence of MTT and PMS during 15, 30, 45, 60 and 75 min, at 37 degrees C. Trophozoites killed by heat or cold were used as controls. The formazan produced into the cells was extracted with isopropanol-HCl and the optical density (OD) was measured at 570 nm. There was a linear relation between the number of viable trophozoites and the OD570 (r2 = 0.98). The trophozoites killed by cold or heat did not reduce MTT. The optimal time of incubation with MTT/PMS was 45 min. The MTT/PMS assay for determining viability of E. histolytica trophozoites is reproducible, rapid and easy to perform and it could be useful to study cytotoxicity and susceptibility to drugs.
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The first application of a newly developed fluorescent formazan in flow cytometry is described. The cell surface redox activity of isolated mouse hepatocytes after incubation with the tetrazolium salt forming the new fluorescent formazan and the DNA content after Hoechst staining have been measured simultaneously. 2 parametric distribution patterns have been obtained. This new and sensitive fluorometric technique can be used for automatic measurements of single cells correlating redox activity with other cell parameters.
Diethylstilbestrol (DES) and four derivatives, viz. tetrafluoro-DES, 3'-hydroxy-DES, Z,Z-dienestrol and hexestrol, were examined for their abilities to form superoxide radicals and to induce DNA strand breaks in the presence of horseradish peroxidase/hydrogen peroxide metabolism in a cell-free system. Furthermore, the induction of strand breaks by these compounds was tested in Syrian hamster embryo (SHE) cells in vitro. Formation of superoxide radicals could be demonstrated by reduction of nitro blue tetrazolium for DES but not for its derivatives. With isolated superhelical DNA, induction of strand breaks in the presence of Fe3+ was observed for DES, tetrafluoro-DES and 3'-hydroxy-DES, while hexestrol and Z,Z-dienestrol were ineffective. In SHE cells, alkaline elution technique showed that DNA strand breaks were induced by DES and all derivatives tested, although only at cytotoxic concentrations. It is concluded that DES, under conditions of peroxidative metabolism, can give rise to superoxide generation and DNA strand breaks, and that these events may play a role in the process of DES-induced cell transformation.
Statins [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors] exert cholesterol-independent pleiotropic effects that include anti-thrombotic, anti-inflammatory, and anti-oxidative properties. Here, we examined direct protective effects of atorvastatin on neurones in different cell damage models in vitro. Primary cortical neurones were pre-treated with atorvastatin and then exposed to (i) glutamate, (ii) oxygen-glucose deprivation or (iii) several apoptosis-inducing compounds. Atorvastatin significantly protected from glutamate-induced excitotoxicity as evidenced by propidium iodide staining, nuclear morphology, release of lactate dehydrogenase, and mitochondrial tetrazolium metabolism, but not from oxygen-glucose deprivation or apoptotic cell death. This anti-excitototoxic effect was evident with 2-4 days pre-treatment but not with daily administration or shorter-term pre-treatment. The protective properties occurred independently of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition because co-treatment with mevalonate or other isoprenoids did not reverse or attenuate neuroprotection. Atorvastatin attenuated the glutamate-induced increase of intracellular calcium, which was associated with a modulation of NMDA receptor function. Taken together, atorvastatin exerts specific anti-excitotoxic effects independent of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition, which has potential therapeutic implications.
The intracellular reduction of a tetrazolium salt (MTT) to a purple formazan is an indicator of cell viability. The MTT test represents a simple colorimetric method to determine cytotoxicity. The present paper describes a new method for solubilisation of the formazan crystals, which makes the assay simpler, more rapid and more reproducible. Different cell lines show different kinetics of formazan formation stressing the need for individual calibration curves. The clear-cut discrimination between cells resistant and non-resistant against oxazaphosphorines indicates that the assay has predictive value for in vivo drug sensitivities. It is concluded that the MTT test is a useful addition to anticancer compound screening programs.