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PubMedDate not supplied

Indirect cytotoxicity of dental materials: a study with Transwell inserts and the neutral red uptake assay.

A modification of the Transwell insert methodology was evaluated by using the neutral red uptake (NRU) assay in a cytotoxicity test. The Transwell insert methodology was developed to assess the biocompatibility of solid materials used in dentistry and, when initially designed, used the release of radiochromium ((51)Cr) in the cytotoxicity assay. Another aim of this study was to evaluate different exposure regimes with which to assess cytotoxicity. The exposure regimes included: a 1-hour exposure in buffer followed by a 24-hour incubation in growth medium; a 2-hour exposure in buffer followed by a 24-hour incubation in growth medium; a 24-hour exposure in serum-limited medium; and a 24-hour exposure in a serum-sufficient medium. The bioindicator target was the Smulow-Glickman (S-G) human gingival cell line and the biomaterials were dental restoratives. The Transwell insert methodology with the NRU cytotoxicity assay as the cytotoxicity endpoint was effective in differentiating the potencies of the dental restoratives; a 2-hour exposure in buffer and a 24-hour exposure in serum-limited medium were the exposure regimes that most clearly differentiated the test agents according to their potencies. The sequence of cytotoxicity of the dental restoratives to the S-G cells was Vitremer > Ketac-Molar Aplicap > Flow-It.

Cell Death↗
PubMed1985

Photocarcinogenesis by methoxypsoralen, neutral red, proflavine, and long UV radiation.

A study of the photosensitizing effects of 8-methoxypsoralen (MOP), neutral red (NR), and proflavine (PF) on the skin of female Swiss albino mice, strain 955, was carried out using fractionated exposure to long ultraviolet light (300-400 nm) and visible light (tungsten emission). The results (1) confirmed MOP photocarcinogenicity, (2) demonstrated that both NR and PF are photocarcinogens, and, further, (3) showed that the above UV light with 2.6% of fluence at 313 nm is a long-term carcinogenic agent even though the total dose of 313 nm was 100 times less than the minimal UV tumorigenic dose in mice. The tumors were mammary adenocarcinomas, carcinomas of skin appendages, carcino-mixo-sarcomas, lymphomas, and one case of thyroid adenocarcinoma. The implications of the above data regarding the controversy about oncogenic risks in photochemotherapy are discussed.

Acridines↗
PubMed1989

Neutral red changes arginine-induced glucagon and insulin response in the rat.

Rats were treated with neutral red (amino-dimethyl-amino-toluaminozine hydrochloride, NR) twice weekly, 5 ml/kg b.wt.-1 of a 2% solution, for five weeks in order to investigate damage to pancreatic A-cells. After 1-5 weeks of treatment plasma glucagon levels were increased to about two times control values. Plasma glucose was maintained at control values. Following arginine stimulation, 8 micrograms intravenously 100 g b.wt.-1 min.-1 for 30 min., plasma glucagon and insulin concentrations increased 10-fold in normal rats. After NR treatment for 1 and 2 weeks the arginine induced glucagon response was reduced to a two-fold increase, after 5 weeks NR treatment arginine again increased glucagon concentrations 10-fold. There was no change in the 10-fold increase of insulin concentrations from arginine infusion during the NR treatment. Immunohistochemical examination of pancreas demonstrated unchanged number and size of Langerhans' islets. The insulin immunoreactivity of the B-cells appeared constant 0-5 weeks during NR treatment, while glucagon immunoreactivity appeared to lower in the A-cells from animals treated for one or two weeks with NR. After three to five weeks of NR treatment, pancreatic glucagon immunoreactivity appeared restored. We conclude that NR leads to a release of glucagon from pancreatic A-cells, with subsequent hyperglucagonaemia, maintained during repeated administrations. Such repeated administrations lead to a transient "glucagon insufficiency" on top of the hyperglucagonaemia, consisting of restricted ability to further release of glucagon, presumably because of pancreatic A-cell glucagon depletion.

Animals↗
PubMed2006

Neutral red as a probe for confocal laser scanning microscopy studies of plant roots.

BACKGROUND AND AIMS: Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology. METHODS: NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye. KEY RESULTS: The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas. CONCLUSIONS: Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.

Microscopy, Confocal↗
PubMed1993

Neutral red assay for measurement of quantitative vero cell cytotoxicity.

A neutral red assay involving Vero cells was used to quantitate the cytotoxic activity of verotoxins (VT) produced by Escherichia coli and to investigate changes in titer caused by altering the composition of the cell culture medium. Three variations on medium 199 were investigated: one involved supplementing the medium with 5% fetal bovine serum (FBS), a second was the use of serum-free (SF) medium that contained 5% bovine serum albumin and 5 mug of fibronectin per ml, and the third involved the use of 4% Ultroser G, a commercial serum replacement. The level of cytotoxicity varied markedly with the type of VT and with the medium that was used. For VT1, there was no difference in cytotoxicity between medium with 5% FBS and SF medium, but cytotoxicity was reduced more than 100-fold in medium containing Ultroser G compared with cytotoxicity in the other media. For VT2, VT2v, and VTe, there was a slight reduction in cytotoxicity in medium containing 4% Ultroser G and a more marked reduction in SF medium compared with cytotoxicity in medium containing 5% FBS.

Journal Article↗
PubMed2000

Rotational relaxation of neutral red in alkanes: effect of solvent size on probe rotation.

Rotational reorientation times of a polar molecule neutral red (NR) have been measured in n-alkanes using steady-state fluorescence depolarization technique. The rotational dynamics of NR in alkanes is described by the Stokes-Einstein-Debye hydrodynamic theory with slip boundary condition. However, we have observed that as the size of the solvent molecule becomes bigger than the size of the solute molecule, the probe molecule experiences reduced friction and the experimentally measured reorientation times are shorter than those predicted by the hydrodynamic theory. These size effects have been analyzed using quasihydrodynamic theories.

Alkanes↗
PubMedDate not supplied

Relation between responses in the neutral red retention test and the comet assay and life history parameters of Daphnia magna.

Responses of the neutral red retention (NRR) assay as test for lysosomal stability and the comet assay as test for DNA integrity were measured in the water flea, Daphnia magna, and compared with mortality and effects on population growth rate during short- or long-term exposure to seven different toxicants. The NRR test and the comet assay were performed with fresh preparations of pieces of tissue from the digestive tract or with cell preparations from whole daphnias. Five toxicants caused responses of the NRR test or the comet assay after short-term exposure at concentrations below the acute toxicity level. Preliminary results of long-term exposure experiments suggest that these biomarker responses can be related to chronic effects on survival and/or reproduction of D. magna. This type of research should provide the basis for future use of the NRR test and the comet assay as early warning biomarkers for effects of toxicants on Daphnia populations.

Animals↗
PubMed1986

[Effect of the segregation of neutral red, acridine orange and ammonium chloride by L cells (subline LSM) on lysosomal hydrolase activity].

Kinetics of Neutral red (NR) and Acridine orange (AO) uptake by cultured L cells (subline LSM) has been studied. It was found that the uptake of both NR and AO, with their constant concentrations in the medium was characterized as a two-phase process. During 2 hours, these cells concentrated as much as 90% of the total amount of NR and AO taken up during the whole incubation period. The segregation and accumulation of NR, AO as well as NH4Cl took place in lysosomes. NR and AO concentrations within the cells exceed by 600 and 400 times, respectively, those in the medium. NR, AO and NH4+ accumulation in cells resulted in inhibition of the activity of the following lysosomal hydrolases: cathepsins B and D, acid lipase, N-acetyl-beta,D-glucosaminidase, beta-galactosidase, acid phosphatase and galactosyltransferase, the latter being a marker of Golgi apparatus. The effect of lysosomal enzyme activity inhibition on the cell economy, and a possible role of lysosomotropic agents as regulators of the lysosomal apparatus functional activity are discussed.

Acridine Orange↗
PubMed1979

[Sorption of neutral red by actomyosin threads during contraction and heating].

Studies of the equilibrium distribution of the cation dye-neutral red by actomyosin threads allowed to establish the fact that different concentrations of dye reveal the existence of different bonds with contractile proteins. Firm bonds (7.3 ccal/M) are discovered at low dye concentrations, and weaker bonds (4.5 ccal/M) are seen at higher concentrations. The contraction of actomyosin threads evoked by ATP or heating is accompanied with similar changes, i. e. by the diminishing of the maximum dye sorption within the range of low dye concentrations and by the increase in the maximum dye sorption within the range of high concentrations.

Actomyosin↗
PubMed2000

Studies on the radiolytically produced transients of neutral red: triplet and reduced radicals.

The spectral and kinetic properties of reduced radicals and the triplet state of neutral red (NR), a phenazine-based dye, have been investigated using pulse radiolysis technique. A mixed water-isopropanol-acetone solvent has been used to study the reduced radicals of NR for a wide pH range of about 1-13, due to limitation of solubility of the dye in aqueous solutions particularly above pH 8. From pH-dependent absorption studies it has been established that the reduced radicals of NR can exist in four different prototropic forms in solution. Three pKa values for the corresponding prototropic equilibria have been estimated. The formation and decay rate constants of reduced radicals have also been measured. The triplet state characteristics of the dye have been investigated in neat benzene solutions, both in the presence and in the absence of triplet sensitizers. The T1-->Tn absorption spectrum and decay kinetics of the triplet state have been measured. The triplet state energy (ET) of NR in benzene have been estimated to be within 36-42 kcal mol-1, using an energy transfer method.

Free Radicals↗
PubMedDate not supplied

Toxicity determined in vitro by morphological alterations and neutral red absorption.

A method is described which combines the use of a visual morphological cytotoxicity assay with a quantitative neutral red (NR) spectrophotometric test, for the assessment of the effect of toxic agents on 3T3 cells in culture. These sensitive and reproducible assays lend themselves to a screening procedure of potential toxicants which can help reduce the use of animals for toxicity testing.

Animals↗
PubMed1992

Quantification of neutral red pinocytosis by small numbers of adherent cells: comparative studies.

A colorimetric assay has been developed for studies on neutral red (NR) pinocytosis by small numbers (below 2 x 10(5)) of adherent cells cultured in 96 well plates. The NR uptake per cell mass was much higher in the sea urchin perivisceral adherent cells and human HL-60 cell line monolayers than in the murine and Atlantic salmon macrophages. The apparent difference points to the usefulness of this novel assay in comparative studies.

Animals↗
PubMed1976

[A cytophotometric study of granules forming in frog erythrocytes during incubation in solutions of neutral red and novocaine].

Granules arising in the cytoplasm of Rana temporaria erythrocytes incubated in solutions containing either a stain (neutral red) or an alcaloid (novocaini) were studied cytophotometrically. The stuff concentrations in the granules were estimated under various conditions of incubations. The stuff concentrations in the granules appeared to be dozens- or hundreds--fold higher than in the incubation solutions. The average concentration in the granules did not depend either on the initial concentration of the stuff solution, or on the incubation time or on the volume of the incubation solution. The stuff concentration in the granule decreases with the increase in the granule size.

Animals↗
PubMed1989

Determination of intracellular pH in the in vitro hippocampal slice preparation by transillumination spectrophotometry of neutral red.

A method is presented for measuring intracellular pH (pHi) spectrophotometrically in hippocampal slices using the pH dye indicator, Neutral red (NR). Measurements of pHi by NR were compared directly with the creatine kinase (CK) equilibrium method. Slices were bathed in artificial cerebrospinal fluid buffered with bicarbonate/CO2. Intracellular pH in hippocampal slices was found to be more alkaline (approximately 0.3 pH units) than buffer pH and far more alkaline (0.5 pH units) than extracellular pH. Resting intracellular alkalinity was observed by using both the NR and the CK equilibrium methods. The method may be useful for studies of pH regulation in intact functioning tissues in vitro where rapid and repeated measurements are necessary or where cell size precludes measurements with pH-sensitive microelectrodes.

Adenosine Triphosphate↗
PubMed1988

[Electron microscopic and biochemical research on the vacuoles formed in the erythrocytes of frogs as affected by neutral red and novocaine].

No lysosomes were found in the frog intact erythrocytes with electron microscope. Under the influence of neutral red (NR-8.7.10(-5) M) and novocaine (N-4.6.10(-3) M) segregation zones (vacuoles) including these substances are formed. Using electron microscopy and morphometry the action of NR and N for 5 minutes up to 48 hours was found to provoke the formation of four types of vacuoles differing in their morphology: with electron-transparent content, with amorphous inclusions and membrane whorls. The dynamics of vacuole formation, of their changes and amount were followed depending on the time of exposition of these substances. Biochemical investigation of both NR and N isolated vacuoles showed in these some activities of lysosomal marker enzymes--acid phosphatase and N-acetyl-beta,D-glucosaminidase. Ultrastructural investigation of acid phosphatase localization in the isolated vacuoles revealed the histochemical reaction product mainly in electron-translucent vacuoles (primary lysosomes) and partly in electron dense ones (secondary lysosomes). On the ground of the above studies a conclusion is made that in frog erythrocytes treated with NR and N lysosome formation is induced to be followed by the induced autophagocytosis and heterophagocytosis. Some possible ways of the vacuolar system formation in frog erythrocytes and the origin of lysosomal hydrolases are discussed.

Animals↗
PubMed1980

The effects of short- and long-term exposure of chick embryos to neutral red on the frequency of sister-chromatid exchange.

The chick embryo was used to study the effects of neutral red (NR) on the frequency of sister-chromatid exchange (SCE) in specific tissues exposed to this mutagen for short and long periods as development proceeded. In short-term trials, aqueous NR at doses of 10, 25 and 100 micrograms was injected in 3-day and 6-day embryos. In each case, embryos were also treated with 5-bromodeoxyuridine (BrdU) for a 24-h period (two cell cycles) and harvested at 4 days and 7 days, resp. A long-term exposure (about 8 cell cycles) was achieved by exposing embryos to NR from day 3 to day 7 of incubation. At a NR dose of 25 micrograms, the chronic exposure resulted in a doubling of the rate of SCE (11.4/cell) over that observed in embryos exposed for only 24 h at either days 3-4 (6.0/cell) or days 6-7 (6.0/cell). At 100 micrograms of NR, the same relationship held with SCE rates of 14.2/cell for the chronic exposure versus rates of 8.0/cell (3-4 days) and 6.9/cell (6-7 days). At 10 micrograms of NR, no such accumulation of SCE occurred upon long-term treatment. These results show an enhanced SCE response upon growth of embryonic cells in the presence of NR for several days. This may be the result of the persistence of past lesions with the addition of more lesions upon continued exposure to NR.

Animals↗
PubMed1990

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes.

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.

Aflatoxin B1↗