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PubMed1987

Differential uptake of neutral red by macrophages from three species of estuarine fish.

Macrophage endocytosis is part of the immune response to foreign material and includes the processes of phagocytosis, ingestion of particulate material, and pinocytosis, the uptake of liquid droplets. This work reports the development of a quantitative method to measure in vitro uptake of neutral red by fish macrophages, based on the spectrophotometric measurement of ingested neutral red dye. We have used this method to monitor the rates of uptake in three species of fish: spot, Leiostomus xanthurus; hogchoker, Trinectes maculatus; and summer flounder, Paralichthys dentatus. For the species tested, there appeared to be an intrinsic pattern of uptake which was highest in flounder, lowest in hogchoker and intermediate in spot. The excellent reproducibility of this assay should make it useful for detecting toxicological effects in the environment.

Animals↗
PubMedDate not supplied

Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity.

The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells. The adhered cells were seeded and then treated and the adhering cells were treated simultaneously upon seeding. Five of the 44 test chemicals were twofold more toxic in adhering cells; ethylene glycol was 28-fold more toxic and mercuric chloride was 5.2-fold more toxic than in adhered cells. The cytotoxicity of dithiothreitol was altered in the same way as that of ethylene glycol, probably by interacting with calcium. When the neutral red uptake inhibition was compared with human toxicity, the correlation coefficient for adhering cells was almost identical to that obtained previously in human hepatoma-derived Hep G2 cells and slightly higher for adhered cells. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. An obviously better correlation was obtained when the strong intoxicant mercuric chloride was withdrawn from the comparison, both for the adhered and the adhering cells. Altogether, the results can be integrated very well with the basal cytotoxicity concept.

Animals↗
PubMed1993

Quantification of adherent and nonadherent cells cultured in 96-well plates using the supravital stain neutral red.

In this study we present a rapid, simple, sensitive, inexpensive, and environment-friendly assay for determination of the number of adherent or nonadherent cells cultured in 96-well plates using the supravital stain neutral red. We describe a validation of the method and demonstrate its application to study the effects of hormones (i.e., parathyroid hormone) and cytokines (i.e., tumor necrosis factor-alpha) on the growth of primary cultures of adherent osteoblast-like cells. In addition we show that this method can also be applied to conveniently determine proliferation of cells which grow in suspension, like the CTLL-2 and B-9 cells, which are widely used to measure IL-2 and IL-6 bioactivity, respectively. In these bioassays the changes in optical density induced by IL-2 and IL-6 measured with the neutral red assay are directly comparable with the relative changes measured with the [3H]thymidine incorporation assay. For all types of cells tested, the optical density at 550 nm was directly proportional to the number of cells. The assay, which can be used for different purposes, is an excellent alternative to already existing methods. It is not only easy to perform but also very reproducible, making it ideal for screening of large numbers of samples. Therefore this assay offers a reliable and flexible tool to determine both stimulatory and inhibitory effects of hormones, cytokines, and drugs on cell growth or to study the effects on cell viability.

Animals↗
PubMed1987

The oxygenation-linked binding of neutral red to spiny lobster hemocyanin. A structural study of the partially oxygenated protein.

The structural change of lobster hemocyanin in cooperative O2 binding was studied by the dye-binding method. It was found that neutral red shows an O2-linked binding to hemocyanin with a higher affinity for the oxy form. The number of the dye-binding sites was estimated to be three in the hexameric molecule of oxyhemocyanin. The course of the structural change in the partially oxygenated hemocyanin was examined using the absorbance change of the bound dye as a measure. It was found that the fractional change in the dye binding was considerably greater than the degree of O2 saturation of hemocyanin. The three-state allosteric model, which was proposed for explanation of the O2 binding properties of lobster hemocyanin [N. Makino (1986) Eur. J. Biochem. 154, 49--55], was also consistent with the effects of the dye on the O2 binding to the native hemocyanin. On the basis of this model, the dye binding to partially oxygenated hemocyanin could be connected with the populations of the affinity states. It was inferred that the binding of neutral red reflects the quaternary structure of the protein. In contrast, O2 binding to the stripped (EDTA-treated) hemocyanin showed a considerable decrease in the cooperativity in the presence of the dye. The O2-binding isotherms could not be explained by the three-state model. It is suggested that the subunit interaction is partially blocked by the dye in the absence of divalent cations.

Allosteric Site↗
PubMed1983

The use of the dye neutral red as a specific, non-toxic, intra-vital stain of islets of Langerhans.

The development of improved methods of islet isolation from the pancreas is hampered by difficulty in identification of islets. A method described by Bensley in 1911 uses neutral red to stain the islets selectively. In the present study the method has been simplified and selective staining of islets in the rat, dog and pig demonstrated by use of light and electron microscopy. Staining of islets with neutral red does not appear to affect their viability as judged by insulin secretion and transplantation in rats.

Animals↗
PubMed2003

Adsorption of arsenate from water using neutralized red mud.

The potential for using seawater-neutralized red mud (Bauxsol), a waste from aluminum manufacturing, as an adsorbent for removing As(V) (arsenate) from water is studied. Herein, adsorption characteristics are investigated and it is shown that adsorption follows the Langmuir model, with the adsorption constants indicating the feasibility of the process. Furthermore, the adsorption is found to increase with decreasing pH (i.e., ligand-like adsorption), higher adsorbent dosages, and lower initial arsenate concentrations. The effects of ions are also tested and it is shown that the adsorption of arsenate decreases in the presence of HCO3-, while Cl- has little effect, and Ca2+ increases the adsorption. Water quality assessment after treatment with Bauxsol indicates that none of the trace elements tested are released from the adsorbent. A TCLP leaching test also reveals that the used adsorbent is not toxic. It is foreseen that Bauxsol may be developed into an efficient low-cost adsorbent for (pre-) treating arsenate contaminated waters.

Journal Article↗
PubMed2004

Adsorption of arsenic from water using activated neutralized red mud.

In this paper activated seawater-neutralized red mud, herein referred to as activated Bauxsol (AB), is used as a novel adsorbent for removing inorganic arsenic (As) from water. The adsorption of As onto AB is studied as a function of contact time, particle size, pH, initial As concentration, AB dosage, and temperature. Kinetic data indicate that the process pseudoequilibrates in 3 and 6 h for As(V) (arsenate) and As(III) (arsenite), respectively, and follows a pseudo-first-order rate expression. Within the range tested, the optimal pH for As(V) adsorption is 4.5, and close to 100% removal can be achieved irrespective of the initial As(V) concentration. Desorption of As(V) is greatest at pH 11.6 where a maximum of 40% can be achieved. In contrast, the optimum pH for As(III) removal is 8.5, and the removal efficiency changes with the initial As(III) concentration. The adsorption data fit the Langmuir isotherm and its linearized form well, with thermodynamic data indicating the spontaneous and endothermic nature of the process. The FITEQL (V.4) and PHREEQC (V.2) computer programs are used to predict As(V) adsorption at various pH values (based on diffuse double layer models). The modeling results fit the experimental results very well and indicate that surface complexation modeling is useful in describing the complex AB surface during the adsorption process. This study shows that As(III) needs to be oxidized to As(V) for a favorable removal using AB and that AB can be a very efficient unconventional adsorbent for removing As(V) from water.

Adsorption↗
PubMed1990

[Reconstitution of the cholesterol-hydroxylating system from adrenal cortex mitochondria by cytochrome P-450scc, adrenodoxin and the redox-mediator neutral red].

It has been found that 3-amino-7-dimethylamino-2-methylphenazine (neutral red, NR) is responsible for the electron transport from the cathode to adrenodoxin (Ad) and cytochrome P-450 (P-450scc) from adrenal cortex mitochondria inaccessible to direct electrochemical reduction under native conditions. The rate constant for Ad reduction by this mediator is equal to 1.1 x 10(5) M-1 s-1 at 25 degrees C; the values of enthalpy and entropy for the activation reaction are 26.6 kJ.mol-1 and -59.6 J.mol-1 deg-1, respectively. Using the shunted electron transport chain NR----Ad----P-450scc, the cholesterol conversion into pregnenolone in an electrochemical cell was performed. Pregnenolone was found to be the sole steroid product of this reaction. Superoxide dismutase and catalase had no effect on the activity of the shunted system. After removal or substitution of Ad the apo-Ad hemoprotein was reduced in a non-productive manner. Under identical reconstitution conditions methylviologen was ineffective as an electron carrier.

Adrenal Cortex↗
PubMed1988

Neutral-red uptake and expression of monocytic antigens in amniotic-fluid mononuclear phagocytes: evaluation of a novel approach for prenatal diagnosis of neural-tube defects.

In cases of fetal neural-tube defects macrophages are present in the amniotic fluid. We found that these viable phagocytic cells take up neutral-red and are easily identified as "red cells" by microscopic examination. This method is suitable for the rapid identification and counting of amniotic-fluid macrophages in suspension. We have studied 298 amniotic fluid samples. In the 226 normal cases studied, 0 to 1,200 macrophages per milliliter amniotic fluid have been found. In contrast, we found 1,250 to 99,000 macrophages per milliliter amniotic fluid in our 70 open neural tube defect (ONTD) cases. Statistical evaluation was performed to estimate the normal and pathologic ranges. Specificity and sensitivity of the neutral-red test and predictive value of positive and negative results have been calculated and presented in comparison with alpha-fetoprotein (AFP) determinations and ultrasonic methods. In 5 cases of anencephaly and 7 normal cases amniotic fluid cells were studied by immunocytochemistry: mononuclear cells present in the abnormal cases showed intense immunoreactivity for the Mo1 and Mo2 surface antigens of the phagocytic cell lineage.

Amniotic Fluid↗
PubMed2003

The effect of lead-contaminated soil from Canadian prairie skeet ranges on the neutral red retention assay and fecundity in the earthworm Eisenia fetida.

The sublethal effects of lead (Pb) on the earthworm Eisenia fetida were evaluated in the laboratory using freshly spiked soil and soil collected from Canadian prairie skeet ranges. After a four-week exposure to soil spiked with lead acetate, earthworm neutral red retention time (NRRT). soil Pb concentrations, and earthworm Pb body burdens were measured. Lysosomal NRRT was reduced in a concentration-dependent manner (p < 0.0001), and NRRT was negatively correlated with earthworm Pb body burdens (r = -0.80, p < 0.0001). To evaluate the effects of aged Pb, earthworms were exposed to soil from three skeet ranges, and responses were compared with three matched reference sites. After a four-week exposure, NRRT, growth, fecundity, soil total Pb levels, and earthworm Pb body burdens were measured. The potentially bioavailable fraction of Pb in these sites was measured using a Ca(NO3)2 extraction. Growth and fecundity did not differ significantly between any of the skeet ranges and their reference sites. However, NRRT was significantly reduced in all three ranges compared with their respective reference sites (p < 0.05), indicating that the neutral red retention assay (NRRA) may be useful for detecting toxicity and potential hazards at Pb-contaminated sites. Lysosomal NRRT was negatively correlated with soil Ca(NO3)2-extractable Pb (r = -0.80, p < 0.0001) and soil total Pb (r = -0.73, p = 0.001). Lysosomal NRRT was negatively correlated (r = -0.67, p < 0.002) with earthworm Pb tissue levels.

Animals↗
PubMed1991

In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model.

A new three-dimensional human skin model consisting of several layers of actively dividing and metabolically active human neonatal foreskin-derived fibroblasts and epidermal keratinocytes grown on nylon mesh has been used to assess the in vitro toxicity of test agents from various classes. Utilizing a slight modification of the published neutral red viability assay for endpoint determination, we have assayed and obtained dose-dependent toxicity curves for test agents from the following classes: detergents (n = 15), alcohols (n = 5), metal chlorides (n = 10), perfumes and colognes (n = 5), shampoos (n = 4), conditioners (n = 3), moisturizers (n = 3), pesticides (n = 3), and antimicrobial preservatives (n = 4). Limited comparisons to in vivo ocular irritancy data with alcohols and detergents are encouraging. We have demonstrated the utility of this metabolically active dermal substrate containing naturally secreted collagen and other extracellular matrix proteins along with the neutral red viability assay for assessing the toxicity of a number of test agents from a variety of different classes with broad industrial applications.

Alcohols↗
PubMed1980

[Effect of neutral red on the cytotoxic activity of olivomycin].

Olivomycin accumulation in living cells first of all occurs in the cytoplasm or probably the cell lysosomes. The nuclear fluorescence of the cells incubated in olivomycin solution was observed in 60-90 minutes. Simultaneously with the nuclear fluorescence there was observed disappearance of the cytoplasm fluorescence. Intensive nuclear fluorescence was recorded in 15-30 minutes in 60-70% of the cells incubated in a solution of neutral red and subsequently treated with olivomycin. The amount of olivomycin bound to the nuclear DNA in dead cells in the presence of Mg++ and at pH 7.2 was so high that the cell nuclei produced yellow-green fluorescence. No binding of olivomycin to the nuclear DNA was observed in acid media. The cytotoxic activity of olivomycin after incubation with the human fibroblasts in a solution of neutral red increased 100 times. Olivomycin may be used for determination of vital activity of the cells when their lysosome function is not impaired.

Cell Survival↗
PubMed1977

The 2-deoxy-D-glucose-neutral red test and vagotomy: an experimental study.

The 2-deoxy-D-glucose - neutral red test proved to be successful in assessing completeness of vagotomy in the baboon both intra- and postoperatively. There were no deleterious side-effects and we found that both products could be sterilized adequately. So far we have used this test of 5 patients in the immediate postoperative phase; in 1 patients, on whom the surgeon thought he had done an incomplete parietal cell vagotomy, the test was found to be positive within a week of the operation.

Alkaline Phosphatase↗
PubMed2001

[Observation of tinea unguium by neutral red staining].

To evaluate the viability of dermatophytes in tinea unguium, we applied neutral red staining (NRS) for nail powder obtained from direct microscopic examination (DME) of positive tinea unguium patients. The nail powder from tinea unguium patients by grinder was applied following three sets of examinations, NRS, DME, and culture on Mycosel medium. The positive rates of nail powder obtained from 50 tinea unguium patients were 35 (70%), 37 (74%) and 2 (4%), respectively. Correlation of three sets of examinations were as follows: twenty-five patients were NRS positive, DME positive and culture negative, 10 patients were NRS negative, DME positive and culture negative, and 8 patients were NRS positive, DME negative and culture negative. These findings suggest that application of NRS for nail powder from tinea unguium patients is a practical method for evaluation of the viability of fungal elements.

Adult↗
PubMed1987

Candidacidal activity of macrophages from three mouse strains as demonstrated by a new method: neutral red staining.

A new method for assessing the candidacidal activity of macrophages utilizing neutral red stain gave results comparable to well established vital staining methods, methylene blue and acridine orange, and to the absence of germ tube formation of Candida albicans. Dead yeast cells are uniformly stained red while viable yeast cells are unstained except for a red stained vacuole. This assay was used to demonstrate the difference in candidacidal activity between resident and in vivo stimulated peritoneal macrophages of CBA/J, BALB/c and CFW mice. Stimulation of peritoneal macrophages with Concanavalin A in vivo resulted in a significant increase in candidacidal activity in the macrophages from male and female mice. In addition, Concanavalin A-elicited macrophages from female BALB/c and CFW mice exhibited greater candidacidal activity than macrophages from male BALB/c and CFW mice whereas Concanavalin A-elicited macrophages from CBA/J male and female mice exhibited a similar candidacidal ability. Resident peritoneal macrophages from male mice were significantly less candidacidal than were macrophages from female mice with the exception of the macrophages from male CBA/J mice which were as active as the macrophages from female mice.

Animals↗
PubMed1999

Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production.

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.

Actinobacillus↗
PubMed2006

Host-guest complexation of neutral red with macrocyclic host molecules: contrasting pK(a) shifts and binding affinities for cucurbit[7]uril and beta-cyclodextrin.

The photophysical properties of the phenazine-based dye neutral red were investigated in aqueous solution in the presence of the macrocyclic host molecule cucurbit[7]uril (CB7) using ground-state absorption as well as steady-state and time-resolved fluorescence measurements. The results are contrasted to those previously obtained for beta-cyclodextrin (beta-CD; Singh et al. J. Phys. Chem. A 2004, 108, 1465). Both the neutral (NR) and cationic (NRH+) forms of the dye formed inclusion complexes with CB7, with the larger binding constant for the latter (K = 6.5 x 10(3) M(-1) versus 6.0 x 10(5) M(-1)). This result differed from that for beta-CD, where only the neutral form of the dye was reported to undergo sizable inclusion complex formation. From the difference in binding constants and the pK(a) value of protonated neutral red in the absence of CB7 (6.8), an increased pK(a) value of the dye when complexed by CB7 was projected (approximately 8.8). This shift differed again from the behavior of the dye with beta-CD, where a decreased pK(a) value (ca. 6.1) was reported. The photophysical properties of both NR and NRH+ forms showed significant changes in the presence of CB7. Fluorescence anisotropy studies indicated that the inclusion complexes of both forms of the dye rotate as a whole, giving rotational relaxation times much larger than that expected for the free dye in aqueous solution. The thermodynamic parameters for the NRH+.CB7 complex were investigated in temperature-dependent binding studies, suggesting an entropic driving force for complexation related to desolvation of the cation and the removal of high-energy water molecules from the CB7 cavity.

Journal Article↗