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Mechanism of stimulation of pinocytosis by trypan blue.

Trypan blue at 50 microgram/ml stimulates the pinocytic uptake of 125I-labelled PVP, but not of colloidal 198Au or formaldehyde-denatured 125I-labelled bovine serum albumin, by the 17.5-day rat visceral yolk sac incubated in vitro. Neither Trypan blue nor a combination of the dye with 125I-labelled PVP stimulated the rate of pinocytosis of liquid by the tissue. Trypan blue itself was shown to enter the yolk-sac cells by adsorptive pinocytosis. It is proposed that an interaction between Trypan blue and 125I-labelled PVP enables the latter substrate to enter the cells adsorptively by so-called 'piggy-back' pinocytosis.

Animals

Abrogation of tumor rejection by trypan blue.

Trypan blue treatment prevented tumor-specific rejection in three animal model systems. These included the spontaneous rejection of tumors of mice (UVT-2051) and guinea pig (line 1 hepatoma) as well as vaccine-induced rejection of a guinea pig tumor (line 10 hepatoma). Secondary immune reactions to line 10 cell challenges were not abolished by trypan blue treatment. Although trypan blue is a potent inhibitor of macrophage cytotoxicity in vitro, the mechanism by which it inhibited tumor-specific rejection has not been established.

Animals

Effect of Evans blue and trypan blue on syncytia formation and infectivity of human immunodeficiency virus type I and type II in vitro.

Polyanionic compounds were used to inhibit infectivity of human immunodeficiency virus in vitro. Suramin, Evans blue, and Trypan blue were shown to inhibit syncytia formation normally observed when HIV-1-infected cells are cocultured with CD4+ cells. The inhibition was more pronounced with Evans blue than with any of the other polyanions studied. The inhibitory effect was significantly weaker in HIV-2 systems. However, the reverse transcriptase activities of both types of viruses were inhibited by Evans blue. Another polyanionic compound, phosphorothioate 28-mer cytidine homopolymer (SdC28) was shown to inhibit syncytium formation induced by HIV-1-and HIV-2-infected cells in an identical manner. Evans blue showed partial blocking of gp120 binding to CD4 in a solid-phase enzyme-linked immunosorbent assay (ELISA). These results suggest that the polyanionic dyes may exert their antiviral effects, at least in part, by interfering with the binding and fusion of HIV with susceptible T cells.

Antiviral Agents

Effects of trypan blue on thyroid secretion: localization of trypan blue within the colloid space and phagolysosomes of thyroid follicles.

Trypan blue was previously shown to directly inhibit thyroid secretion following TSH stimulation. Inhibition of both colloid droplet formation and thyroglobulin proteolysis was demonstrated. By observing the characteristic bright red fluorescence of the dye-protein complex, we have demonstrated that trypan blue rapidly enters the colloid space and combines with thyroglobulin. In addition, the dye in association with thyroglobulin has been demonstrated within phagosomes and phagolysosomes by centrifugation of the lysosomal (P15) fraction on both sucrose and Percoll density gradients. Lability or latency of the dye with the phagolysosomal contents was demonstrated and the dye was found in association with thyroglobulin by column chromatography. It is proposed that the complexing of trypan blue to thyroglobulin alters its attachment to specific follicular cell receptors, inhibits pinocytosis, and, thus, thyroid hormone secretion.

Animals

Direct effects of trypan blue on thyroid secretion.

Trypan blue directly inhibited in vitro thyroid secretion (butanol soluble 125I release to the media) induced by both thyroid stimulating hormone (TSH) and dibutyryl cAMP. Intracellular colloid droplet counts were also decreased. Inhibition was directly proportional to dye concentration and could be overcome by supramaximal TSH and dibutyryl cAMP. Inhibition could be observed as early as 20 min of incubation, was not increased by preincubation, and could even be demonstrated after TSH in vivo. Trypan blue, in vivo, produced similar inhibition of thyroid secretion. Incubation of 125I-thyroglobulin with lysosomal enzymes revealed inhibition with much lower concentrations of dye. Inhibition of lysosomal enzyme(s) would not appear to explain the marked decreases in colloid droplets, and this may represent two separate effects of trypan blue on thyroid secretion.

Animals

Inactivation of measles and herpes simplex viruses by trypan blue.

The dye trypan blue inhibits infection of cells in tissue culture by measles and herpes simplex viruses at concentrations in excess of 0.01% (w/v) principally by direct inactivation of the virions. The inactivation is temperature-dependent (10(4) times greater at 37 degrees C than at 4 degrees C) and its mechanism appears to include inhibition of virus adsorption to cells.

Animals

Accumulation of DNA, RNA, and protein by cultured rat embryos following maternal administration of a teratogenic dose of trypan blue.

The effect of trypan blue on the rates of DNA, RNA, and protein accumulation in rat embryos was investigated using a system of whole embryo culture. Embryos were isolated on the 10th gestational day (sperm day = Day 0) either 4 or 24 hr after maternal injection. The cultivation period was for 24 or 42 hr. The rate of DNA and RNA accumulation in the embryo following either a 4- or 24-hr previous maternal treatment was significantly decreased at at both 24 and 42 hr of cultivation. Protein values were significantly reduced after a 4-hr maternal exposure, but only slightly reduced after a 24-hr maternal exposure. No significant differences in DNA, RNA, or protein content were observed at the beginning of cultivation, following either a 4- or 24-hr previous maternal treatment. The percentage of embryos establishing a visceral yolk sac circulation was greatly reduced, and this supports the conclusion that trypan blue interferes with yolk sac function. The most obvious teratogenic insults include failure of neuropore closure, retention of a dorsiflexed position, and cranial edema.

Animals

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro.

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.

Abnormalities, Drug-Induced

Direct effects of trypan blue on cardiac extracellular macromolecule synthesis.

The direct effects of the cardiac teratogen trypan blue on some parameters of embryonic chick heart metabolism were examined in vitro. Trypan blue inhibits chondroitin sulfate biosynthesis but stimulates incorporation of 3H-glucosamine into hyaluronate, heparin, and glycopeptides. 3H-fucose incorporation into glycoprotein is also stimulated. Total incorporation of 14C-labeled amino acids is elevated in the presence of trypan blue. Trypan blue does not cause qualitative changes in labeled end products; rather the metabolic alterations are quantitative. The possible exceptions are glucosamine-labeled glycoproteins since quantitative differences in glycopeptides could reflect qualitative differences in parent glycoproteins. These observations suggest that abnormal heart development, induced by trypan blue, may result from a multiplicity of metabolic alterations and not from any single chemical change.

Animals

Effects of trypan blue treatment on the immune responses of mice.

It has been reported that trypan blue treatment decreases the nonspecific resistance of mice to transplanted tumors and inhibits the in vitro cytotoxic activity of activated macrophages. We wished to determine whether this effect of trypan blue could be due to a selective inhibition of certain macrophage functions or whether it reflected a broader form of immunosuppression. We therefore tested the effects of trypan blue on a variety of immunological responses. Treatment of mice with trypan blue delayed their rejection of skin allografts and transplants of a highly antigenic syngeneic ultraviolet light-induced tumor. Trypan blue treatment of either donor or recipient decreased the local graft-versus-host reaction. Filtration of lymph node cells from trypan blue-treated donors on a nylon wool column before use in the graft-versus-host assay abrogated the depressive effect of trypan blue. A transient reduction in the blastogenic response of spleen cells to concanavalin A and lipopolysaccharide mitogens was observed after a single injection of trypan blue, but the response of lymph node cells was unaffected. The depressed response of splenic lymphocytes was not entirely reversed by removal of adherent cells. The primary and secondary hemagglutinin responses to sheep erythrocytes were unaffected in trypan blue-treated mice, and the proportion and phagocytic activity of thioglycolate-induced peritoneal macrophages were also unaltered. We conclude that treatment of mice with trypan blue selectively inhibits certain macrophage functions but, at high doses, it can also inhibit some lymphocyte activities.

Agglutinins

Comparative pharmacokinetics of trypan blue in female Sprague-Dawley and Long-Evans rats.

Administration of trypan blue to pregnant Sprague-Dawley or Long-Evans rats, during an identical stage of embryogenesis, has been associated with malformations in 97 and 17% of the offspring, respectively (Gunberg, Anat. Rec. 1958, 130, 310). In the present study the comparative pharmacokinetics of trypan blue in the two strains were investigated. Female Sprague-Dawley and Long-Evans rats were injected sc with 10 mg trypan blue/rat [0.5 ml 2% (w/v) trypan blue in distilled water]. Blood samples were collected from the post-orbital plexus at times ranging from 5 min to 480 hr after dosing. The peak serum concentration of trypan blue was greater in Sprague-Dawley rats. Pharmacokinetic analyses of concentrations of trypan blue in serum resulted in fitting a two-compartment open model, with first order absorption, for both strains. Elimination of trypan blue from the central compartment was faster in Sprague-Dawley rats. This phenomenon was associated with a net movement out of the central compartment, predicted by the ratio of intercompartment rate constants. It is possible that the reported differences between the two strains in the teratogenic effects of trypan blue could be attributable, in part, to these observed pharmacokinetic dissimilarities.

Absorption

Trypan blue inhibits complement-mediated phagocytosis by human polymorphonuclear leukocytes.

Trypan blue completely inhibited attachment of human polymorphonuclear leukocytes (PMN) to Sepharose beads coated with C3 ant to sheep erythrocytes coated with IgM plus C3, but it did not inhibit attachment to erythrocytes coated with IgG. These results suggested that trypan blue inhibited C-mediated attachment to PMN membranes. Corroborative studies were performed with a strain of Staphylococcus aureus that requires C but not antibody, for opsonization and that activates the alternative pathway. Trypan blue was not toxic to PMN or bacteria, did nto interfere with immunoglobulin or C interactions, and did not affect attachment of opsonins to bacteria. However, the dye impaired PMN killing of S. aureus in normal nonimmune serum by inhibiting bacterial attachment to and ingestion by PMN. Further evidence that the inhibition was at the C3 receptor level came from the observations that, 1) once staphylococci were attached to PMN at either 37 degrees C or 0 degrees C, addition of trypan blue did not inhibit killing; and 2) trypan blue inhibited killing of bacteria opsonized with serum sufficient in C but previously absorbed at 0 degrees C with the same strain of organism to deplete specific antibody. Further studies with this agent may elucidate the roles of opsonic receptors on human phagocytes.

Cell Adhesion

Some additional observations relating to the mechanism of trypan blue induced teratogenesis.

The mechanism and site of teratogenic action of trypan blue on mammalian embryos was reinvestigated. The experiments to be presented include (1) an analysis of the effect of trypan blue treatment on the morphology of the early mouse egg cylinder, (2) a demonstration of the effect of dye treatment on the enzyme acid phosphatase of yolk sac epithelium using histochemical procedures. Results obtained from these experiments indicate that trypan blue injected into mothers on day 7 of gestational age leads, within 12 to 24 hours after treatment, to dramatic abnormalities in 90-95% of egg cylinders examined. The frequency of gross malformations obtained by this treatment is considerably less when litters are examined at later stages of gestation. Acid phosphatase activity in yolk sac epithelium is depressed by the dye treatment, but there is no difference between enzymatically depressed yolk sacs of malformed embryos and yolk sacs surrounding normally appearing litter mates both obtained from trypan blue treated mothers. The hypothesis that trypan blue may exert its teratogenic effect by the direct exposure of egg cylinder stages to the dye, and that some of the egg cylinders affected may subsequently repair, is recommended for further testing.

Abnormalities, Drug-Induced

On the prenatal noxious effects of trypan blue and of a related azo dye.

Since 1948 trypan blue has been a well-known and extensively used experimental teratogen, belonging to the group of azo dyes. Chemically, trypan blue consists of a biphenyl molecule (0-tolidine or benzidine) combined by means of azo linkages with two molecules of a substituted naphthalene. Between 1987-89 the effect of the replacement of the biphenyl molecule by a molecule of p,p'-diaminobenzanilide upon the prenatal noxious action of trypan blue has been controlled. Investigations were carried out on three species: chick embryos, albino rats and albino mice. In the species used, the replacement annihilates the teratogenic properties of the dye, with the persistence of some embryotoxic effects. On the other hand, the control of o-tolidine and of p,p'-diaminobenzanilide revealed that no one had teratogenic properties (only some embryotoxic effect, more marked in the case of o-tolidine). It results that the teratogenic action of trypan blue cannot be attributed to the o-tolidine molecule proper but to an effect which results (in a for the moment unknown manner) from its combination with the other parts of the dye molecule.

Abnormalities, Drug-Induced

[Trypan blue staining capacities of the culture cells (2)--The mechanism of staining].

The cultured cells dispersed by rubber policeman were stained with trypan blue, but these cells were viable at the meaning of keeping viral infective center forming capacity and at the meaning that trypan blue stained cells were transferred to unstained ones by trypsin treatment. This phenomenon may be important not to miscount numbers of the viable cells in cultured cells. Phagocytosis (endocytosis) is well known as a reaction inhibited by NaF or monoiodoacetate, but these metabolic inhibitors did not inhibit trypan blue staining of the cells dispersed both by rubber policeman and 0.05% trypsin treatment. The cells suspended in higher osmotic pressure media were more stainable with trypan blue than lower ones. Addition of K+ to the reaction mixture was not effective on trypan blue staining. These results suggest that "alive" cells dispersed by rubber policeman are stained with trypan blue not to utilize phagocytosis (endocytosis) or Na+-K+ pump, whose reactions couple with energy production. Trypan blue staining capacity of the cultured cells may be depended on pore sizes of protein channels (for example, fixed or gated transport protein channel) in lipid-bilayers of cell membranes. It is concluded that the cells dispersed by trypsin treatment (spherical shape in microscopical observation) and spherical cells in rubber policeman dispersing ones are unstained with trypan blue to contain smaller pore size channels in the cell membranes, but the rest, amoeba-like form cells dispersed by rubber policeman and dead cells are stained to contain larger ones.

Cell Survival

Binding and uptake of trypan blue by developing oocytes of Locusta migratoria migratorioides.

Resorbing oocytes are heavily stained by trypan blue injected into the haemolymph; this serves as a basis for a quick and convenient method for measuring the degree of resorption. Oocytes in the beginning of their development are most susceptible towards resorptive tendencies. The uptake of trypan blue by normally developing oocytes is proportional to the oocyte surface. From 'double-marker' experiments, in which trypan blue is injected into the haemolymph together with [3H]inulin (which does not bind to the oocyte membrane) it is estimated that the contribution of binding in the interiorization of trypan blue is in the order of 80%, under the conditions given. In vitro incubations show the interaction of trypan blue with the membrane to be electrostatic in nature.

Animals

Suppression of cell-mediated tumor cell lysis and complement-induced cytotoxicity by trypan blue.

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.

Animals