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PubMed1981

The failure of glutamic acid to protect the rat embryo against the action of trypan blue.

The effect of L-glutamic acid on the embryolethal and teratogenic action of trypan blue was investigated in Wistar albino rats. L-glutamic acid was either incorporated into the diet, from gestation day 2 to day 20, or suspended in sesame oil and administered by gavage, from gestation day 6 to day 10. The day of finding sperm in the vaginal smear was designated day 0 of pregnancy. A teratogenic dose of trypan blue was injected at day 8 of pregnancy, either intraperitoneally (14 mg/kg maternal body weight) or subcutaneously (160 mg/kg). The amount of glutamic acid consumed, after the injection of trypan blue, ranged from 600 to 1,500 mg/rat/day. Pregnancy was terminated at day 20, and the fetuses were recovered and examined. Glutamic acid failed consistently to protect the rat embryo against the lethal and teratogenic action of trypan blue. These results are in contrast to those obtained in mice. the administration of sesame oil alone was found to cause embryonic death but not malformations.

Abnormalities, Drug-Induced↗
PubMed2002

Determining the lowest trypan blue concentration that satisfactorily stains the anterior capsule.

PURPOSE: To determine the lowest concentration of trypan blue that will stain the anterior capsule satisfactorily to perform a safe continuous curvilinear capsulorhexis and to describe a staining technique using a dispersive viscoelastic material. SETTING: Cerrahpasa School of Medicine, University of Istanbul, Istanbul, Turkey. METHODS: Trypan blue 0.1% (Vision Blue) was diluted by half by adding an equal volume of balanced salt solution (BSS) in a stepwise pattern continuously until the concentration failed to stain the anterior capsule satisfactorily. After the dilution, 4 concentrations of the dye were obtained: 0.050%, 0.025%, 0.0125%, and 0.00625%. The volume of dye used for staining was 0.1 cc at all concentrations. Each concentration of the dye was applied using a classic air-bubble technique or a technique in which the dye was injected under sodium hyaluronate 3.0%-chondroitin sulfate 4.0% (Viscoat) onto the anterior lens surface without using an air bubble to reform the anterior chamber. Each diluted concentration was used in 10 eyes by the same surgeon, with 5 eyes having the air-bubble technique and 5 having the viscoelastic technique. The 0.1% concentration was used in 5 eyes, all having the viscoelastic technique. RESULTS: Trypan blue in concentrations as low as 0.0125% stained the anterior capsule satisfactorily. In addition, staining was possible under the Viscoat. The viscoelastic technique was faster, safer, and easier to perform than the air-bubble technique. CONCLUSIONS: As trypan blue is a potentially carcinogenic vital dye and its possible long-term side effects are unknown, the lowest effective concentration should be used. A concentration lower than 0.1% was effective in staining the anterior capsule even under dispersive viscoelastic material.

Capsulorhexis↗
PubMed1973

The effect of triton WR-1339 on the subcellular distribution of trypan blue and 125 I-labelled albumin in rat liver.

1. The density-gradient distribution patterns of acid phosphatase, Trypan Blue and denatured (125)I-labelled albumin were studied by discontinuous sucrose- and isopycnic sucrose-density-gradient centrifugation on combined heavy and light mitochondrial (M+L) fractions of liver isolated from normal rats and from rats injected with Triton WR-1339. 2. The results obtained from the subfractionation of the M+L pellet of normal animals indicate that the equilibrium density of Trypan Blue and acid-insoluble radioactivity is the same as that for acid phosphatase, which suggests they are bound by a common membrane to form a distinct subcellular population of lysosomal nature. 3. In contrast, the analysis of the isopycnic gradients obtained on subfractionation of M+L pellets of liver isolated from rats treated with Triton WR-1339 show that the acid-insoluble radioactivity has an equilibrium density around 1.21, whereas the acid hydrolases, including cathepsin D, show the characteristic shift to an equilibrium density of around 1.12. Trypan Blue is distributed along the gradient with distinct peaks at densities 1.22 and 1.12. 4. Similar equilibrium-density distribution patterns were obtained with M+L pellets isolated from rats pretreated with Triton WR-1339 but not injected with Trypan Blue. 5. Treatment of the rats with Triton WR-1339 does not affect albumin digestion of isolated intact lysosomes despite the fact that most of the cathepsin D and the albumin ingested by phagocytosis are located in different vacuoles. 6. It is concluded from these experiments that in the liver of animals treated with Triton WR-1339 (125)I-labelled albumin is located within heterophagosomes which do not fuse with heterolysosomes containing the non-ionic detergent Triton WR-1339. The inability of these two lysosomal populations to fuse is not due to Trypan Blue.

Acid Phosphatase↗
PubMed1984

Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture.

Erythrosin B and trypan blue are tested and compared for their effectiveness as vital exclusion stains for mammalian cells in monolayer culture. Both stains are supposed to mark cells that have lost membrane integrity. Fluorescein diacetate (FDA), an efficient vital inclusion stain, is used as a control, as it marks cells retaining membrane integrity. Erythrosin B and FDA are used as fluorescent dyes, whereas trypan blue colors via light absorption. The effectiveness of both vital exclusion stains is assayed by their ability to stain a high percentage of monolayer cells exposed to treatments lethal to an entire cell population. Two types of lethal treatment, severe heat and metabolic poison, are employed. Erythrosin B stains all monolayer cells immediately after complete lethal treatment. Trypan blue optimally stains only about 60% of monolayer cells. Cell staining by erythrosin B and by FDA are found to be mutually exclusive. This result demonstrates the coincidence of viability indications by erythrosin B and FDA and thus confirms the reliability of both viability stains as they probe membrane permeability via independent mechanisms. This study shows that erythrosin B is an effective, nontoxic, and convenient fluorescent vital exclusion dye for three mammalian cell lines in monolayer culture, but tends to disqualify trypan blue for this application.

Animals↗
PubMedDate not supplied

Trypan Blue as a marker of plasma membrane permeability in alloxan-treated mouse islet cells.

Suspensions of pancreatic islet cells from noninbred ob/ob-mice were incubated with Trypan Blue. Microscope photometry showed that apparently viable cells excluded the dye completely, whereas the nuclei of nonviable cells accumulated Trypan Blue by a saturable process. The nucleus-to-medium dye gradient was more then 30:1 in media containing 0.1% or less Trypan Blue. The apparent affinity constant for nuclear binding of the dye was 3.1 X 10(4)l/mol. Albumin partially inhibited the nuclear staining. More than 0.5% Trypan Blue in the medium was toxic per se. In the absence of albumin, 0.5 or 20 mmol/l alloxan, 1 mmol/l N-ethylmaleimide, or 0.1 mmol/l chloromercuribenzene-p-sulphonic acid, but not 20 mmol/l streptozotocin, increased the frequency of islet cells stained with 0.1% Trypan Blue. The absorbance of nuceli was also increased in cells treated with alloxan or N-ethylmaleimide, but not in those treated with chloromercuribenze-p-sulphonic acid. It is concluded that alloxan rapidly increases the permeability of the plasma membrane in mouse beta-cells. This action of alloxan appears to be more acute than any such effect of streptozotocin.

4-Chloromercuribenzenesulfonate↗
PubMed1969

Protein digestion in isolated lysosomes inhibited by intralysosomal trypan blue.

Control rats and rats treated with subcutaneous trypan blue were injected intravenously with denatured albumin-I(125). Lysosome-rich fractions of their livers, when incubated at 22 degrees C in osmotically protected medium (pH 7.4), retained their capacit to digest albumin-I(125). The rate of digestion was lower in suspensions pre-pared from rats treated with trypan blue than in control suspensions, but rates of lysosome breakage were not different. T'hese results and other experimental evidence suggest that trypanblue concentrated within lysosomes can inhibit intralysosomal digestion, probably by inhibition of lysosomal proteinases.

Acid Phosphatase↗
PubMed1977

Alloxan cytotoxicity in vitro. Microscope photometric analyses of Trypan Blue uptake by pancreatic islet cells in suspension.

Suspensions of islet cells were prepared by shaking pancreatic islets from non-inbred ob/ob mice in a Ca2+-free buffer. The cells were incubated with or without 20 mM-alloxan, and subsequently with Trypan Blue. The uptake of Trypan Blue by cell nuclei was analysed by microscope photometry and by counting the frequency of cells appearing stained on visual inspection. Cells classified as stained or unstained by inspection showed no overlap in nuclear absorbance. Suspensions not exposed to alloxan contained 70-80% of unstained cells. Alloxan markedly decreased the frequency of unstained cells, an effect counteracted by 5 or 20 mM-D-glucose. The spectrum of Trypan Blue in islet-cell nuclei was red-shifted by about 20 nm. A similar red-shift was observed on adding the dye to solutions of albumin or histones, but not on mixing the dye with DNA. Binding to basic proteins may explain the concentrative uptake of Trypan Blue in dead cells and contribute to the oncogenic transformation of phagocytotically active cells. Beta-Cells in vitro are killed by alloxan and hence represent a valid model for studying the diabetogenic action of the drug.

Albumins↗
PubMed1984

[Studies on the pharmacological bases of fetal toxicity of drugs. (VI) Teratogenic effects of trypan blue and related compounds in rats].

The teratogenicity of trypan blue and its related compounds was studied in Wistar rats and the following results were obtained: 1) o-Tolidine, 1-amino-8-naphthol-3, 6-disulfonic acid or 1-nitronaphthalene-3, 6-disulfonic acid was injected subcutaneously on day 7 of pregnancy (sperm = day 0). No fetotoxicity was observed in any group. 2) The main fractions, blue fraction (blue fr.) and red fraction (red fr.), were separated from commercial trypan blue (C-TB) by silica gel column chromatography. C-TB, blue fr. or red fr. was injected into pregnant rats subcutaneously on day 7 of pregnancy. The incidence of malformed fetuses after injection of blue fr. was higher than that of C-TB, and the types of malformations induced by C-TB and blue fr. were similar. However, no fetotoxicity was detected after injection of red fr. 3) Blue fr. or red fr. was injected into the exocoelom on day 11 of pregnancy. The incidence of malformed fetuses in the group injected with blue fr. (2.5 micrograms/embryo) was 39%, and the types of malformations were abnormal tail and vertebrae, which were also observed after injection of C-TB or blue fr. into pregnant rats. No significant teratogenic effect was observed after injection of red fr. From these data, it was concluded that the teratogenic effect of C-TB might be due to the blue fr., but not the red fr.

Abnormalities, Drug-Induced↗
PubMed1989

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine.

Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neuronal death, and for estimating the degree of specificity of a toxin.

Animals↗
PubMed1975

Physiological effects of hypoxia and trypan blue in 17-day chick embryos.

Seventeen-day chick embryos were divided into 5 groups and treated as follows: (1) untreated, (2) yolk-sac injected with 0.1 ml of saturated trypan blue solution solution in sterile saline, (3) saline, (4) hypoxia, i.e., 10.5% oxygen, and (5) hypoxia plus trypan blue. After 5 h hypoxia-treated embryos had an increased mortality rate, severe hypoglycemia, reduced blood pH, elevated plasma potassium, and reduced CO2 content. Trypan blue treatment induced few deaths and few physiological imbalances. Hypoxia plus trypan blue was without synergistic effects and had effects that did not differ significantly from hypoxia alone. This lack of response of 17-day chick embryos to high doses of trypan blue may be related to a marked decline in oxygen consumption by the yolk at this age.

Animals↗
PubMed2003

A histological analysis of lens capsules stained with trypan blue for capsulorrhexis in phacoemulsification cataract surgery.

PURPOSE: Staining of anterior lens capsules with dye to facilitate completion of continuous curvilinear capsulorrhexis is now being used more frequently in phacoemulsification of white and mature cataracts with poor red reflexes. This study examined the histological characteristics of anterior lens capsules stained with trypan blue. The layer(s) of the lens capsule that stained with dye and the extent of accumulation of dye in these layers of the lens capsule were determined. To the best of our knowledge this has not been described before. METHODS: A series of 10 stained lens capsules were analysed histologically. The dye used in this study consisted of a standard sterile, noninflammatory, nonpyrogenic, 2 ml solution containing 0.6 mg/ml of trypan blue. Following capsulorrhexis, samples were sent to the laboratory for histological analysis. Frozen sections (8 microm) were prepared and examined with the light microscope. All 10 capsules were cut by frozen section to preserve trypan blue staining (which would be leached by processing) and then subjected to immunohistochemistry for collagen IV. Immunohistochemical analysis using markers for type IV collagen were done on formalin-fixed specimens for morphological comparison with the frozen sections. A counterstain highlighted the epithelium. RESULTS: Continuous curvilinear capsulorrhexis was successfully and easily completed in all cases without any complications. Frozen section analysis using light microscopy demonstrated accumulation of trypan blue dye in the basement membrane of the lens capsule. Staining was concentrated in the portion of the membrane adjacent to the lens epithelium. The lens epithelium could not be clearly identified on the frozen sections. Consequently, immunohistochemical analysis with markers for type IV collagen was performed. A counterstain highlighted the epithelium. This confirmed that the layer staining with trypan blue was the basement membrane, a consistent feature on all the specimens. CONCLUSION: Trypan blue selectively stains the basement membrane of the anterior lens capsule. There is a concentration of dye in the basement membrane adjacent to the lens epithelial cell layer. The lens cortex does not appear clinically to stain with trypan blue. This enables surgeons to distinguish the lens capsule from the cortex and provides sufficient contrast for successful completion of continuous curvilinear capsulorrhexis during cataract surgery.

Basement Membrane↗
PubMed2002

Trypan blue assisted phacoemulsification in corneal opacities.

AIM: To evaluate the efficacy of trypan blue for enhancing visualisation during phacoemulsification and foldable lens implantation in cases of cataract with corneal opacities. METHODS: 11 eyes of 11 patients with nebulomacular corneal opacities involving the visual axis with partially and visually debilitating cataract underwent trypan blue assisted phacoemulsification with foldable intraocular lens implantation. The patients were followed at the first day, first week, first month, and third month postoperatively. Completion of capsulorhexis, phacoemulsification with foldable lens implantation, and postoperative best corrected visual acuity were measured. RESULTS: The dye improved visualisation of the anterior capsule and a complete capsulorhexis could be performed successfully in all eyes. In all but one eye phacoemulsification was accomplished successfully. In one eye with Fuchs' dystrophy penetrating keratoplasty was performed later. Visual acuity was < or = 6/60 preoperatively in all eyes and improved to > or = 6/24 in eight eyes postoperatively. CONCLUSION: Trypan blue assisted phacoemulsification may be performed in selected cases of corneal haze/opacification with cataract. It provides acceptable visual outcome in cases awaiting penetrating keratoplasty or in cases where it is not feasible or promising.

Adult↗
PubMed1988

Lymph node siderosis in trypan blue treated rats.

The accumulation of iron in the lymph nodes of trypan blue treated rats was examined as a possible experimental counterpart of the lymph node siderosis which occurs in patients with Hodgkin's disease. Lymph nodes removed from the hilus of the liver, retrosternal area, axilla and root of the small bowel mesentery were examined histologically for iron in rats receiving 6-20 subcutaneous injections of trypan blue at biweekly intervals and in control rats. An increase in erythrophagocytosis accompanied by a progressive increase in the amount of stainable iron was found in the RE cells of nodes located in the lymphatic outflow tract of the liver. As in patients with Hodgkin's disease, an increase in erythrophagocytosis together with the prolonged retention of iron by RE cells appears to account for the accumulation of iron in the lymph nodes of trypan blue treated rats.

Animals↗
PubMed1976

Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.

Animals↗
PubMed1995

Effect of trypan blue on the activity of lysosomal enzymes, tumor growth and cell ultrastructure in B16 melanotic melanoma in mice.

Trypan blue is known to act as a lysosome membrane destabilizer. We investigated the effect of this dye on the activity of cathepsin D, acid phosphatase and arylsulfatase in tissue homogenates of B16 melanotic melanoma, transplanted subcutaneously in C57BL/6J black male mice. We also examined the tumor growth and the ultrastructure of its cells. The mice were given subcutaneous injections of the suspension of B16 cells (10(6)), and then received the trypan blue solution intraperitoneally in four divided doses, reaching the total does of 0.1 mg/g b.w. (group I) or 0.4 mg/g b.w. (group II). The dye was administered each other day after the tumor transplantation. The control mice were injected with melanoma cells only. The animals were killed 2 weeks after the beginning of the experiment. We found that the activity of lysosome hydrolases was increased by 30% to 50% in groups I and II, respectively, as compared to the control animals. The tumor growth in groups I and II was accelerated, and some ultrastructural changes in the melanoma cells were observed. These included irregular shape of the nucleus, uneven dispersion of the chromatin, increased number of premelanosomes and Golgi structures. The number of lysosomes, however, remained unaltered. We postulate that the trypan blue promotes tumor growth through the enhancement of the activity of lysosomal hydrolases; this may be due to the increased permeability of lysosome membranes caused by the trypan blue.

Animals↗
PubMed1976

Effects of trypan blue on the development of the garden lizard, Calotes versicolor.

The eggs of Calotes versicolor were exposed to different concentrations of trypan blue around the time of appearance of limb buds. The treatment resulted in malformations of limbs, hemorrhages in various locations, microphthalmia, kinky tail, and retarded development and growth of the contained embryos. The proportion of affected embryos varied significantly from clutch to clutch, perhaps due to genetic variation. All treated embryos, including those apparently unaffected, had trypan blue in the blood circulation, but not in the amniotic cavity or extraembryonic celom. Trypan blue was seen in kidney, liver, and other organs in decreasing order of frequency and concentration. On histological examination a change in the mitotic and necrotic frequency of the cells was noticed as one of the early events. The initial necrosis was close to blood vessels. The protective role of yolk sac in trypan blue-induced teratogenesis is discussed and contrary to the widely held view it is concluded that the yolk sac appears to play no such significant role in Calotes. It is suggested that trypan blue directly affects Calotes embryos, perhaps by altering cell permeability.

Abnormalities, Drug-Induced↗
PubMed1978

[Myelopoiesis in lymph nodes of mice treated with trypan blue (author's transl)].

When mice are injected with trypan blue inside the peritoneal cavity immediately after birth, a delay of the lymphocyte colonization and an enhancement of the lymph node myelopoiesis are observed. In normal mice some granulopoiesis takes place in lymph nodes between 4th and 12th days after birth, but after trypan blue treatment granulopoiesis increases and erythroid colonies appear. These findings indicate that the blockade of RES enhances the hemopoiesis in lymph nodes.

Animals↗
PubMed2004

Time course of lens capsule staining using trypan blue and indocyanine green: in vitro study in porcine eyes.

PURPOSE: To determine whether staining of the lens capsule with trypan blue 0.1% and indocyanine green (ICG) 0.5% diminishes with time and whether it differs between the anterior and posterior capsules. SETTING: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan. METHODS: Crystalline lenses removed from porcine eyes were stained for 10 seconds with 0.1 mL of trypan blue 0.1% or indocyanine green 0.5%. They were then placed in distilled water and observed for the persistence of staining over time. In a second experiment, the anterior chamber and internal aspects of the anterior capsule and internal and vitreous aspects of the posterior capsule were gently irrigated with 0.1 mL of trypan blue 0.1% or ICG 0.5%. After 10 seconds, the capsules were irrigated with distilled water and the staining intensities were compared. RESULTS: Staining was not diminished 30 seconds, 5 minutes, or 1 hour after application of either dye. No difference was evident in staining intensity or diminution with time between the anterior and posterior capsules, but the external aspects were stained more than the internal aspects with both dyes. Trypan blue produced more intense staining than ICG. CONCLUSIONS: Since the intensity of capsule staining in the intact lens did not change during a 1-hour immersion in water, capsule dyes may not dissipate fully during cataract surgery. As possible long-term adverse effects have not been ruled out, capsule dyes should be used in a low concentration for a short exposure time.

Animals↗