In vivo liver and lung targeting of adriamycin encapsulated in glutaraldehyde-treated murine erythrocytes

Treatment of adriamycin-loaded erythrocytes from B6D2F1 mice with 0.1% glutaraldehyde produced the following effects: a considerable decrease in the in vitro leakage of the unmodified drug and a selective liver (and, to a lesser extent, lung) uptake of the encapsulated drug (70% of the injected dose) compared to drug leakage from, and tissue distribution of, carrier erythrocytes not treated with glutaraldehyde. The liver vascular bed was not saturated by five daily intravenous injections of 20 microliters of glutaraldehyde-treated erythrocytes, which allows a total dosage of 200 micrograms of the drug (half the LD50 value) to be administered. No appreciable liver damage results from extensive and prolonged uptake of glutaraldehyde-treated carrier erythrocytes. Entrapment of adriamycin within erythrocytes along with glutaraldehyde treatment of the carrier cells seems to be a promising therapeutic strategy against liver (and lung) tumors.     

Encapsulation of interleukin-2 in murine erythrocytes and subsequent deposition in mice receiving a subcutaneous injection

Radiolabeled recombinant human interleukin-2 (IL-2) was successfully encapsulated in both mouse and sheep erythrocytes. Of the added IL-2, 70% was recovered bound to or encapsulated within the carrier cells. Erythrocytes containing IL-2 were stable in vitro and most of the IL-2 remained associated with the cells following a 16-h incubation at 37 degrees C. When carrier erythrocytes containing IL-2 were injected subcutaneously into mice, intact [35S]IL-2 was detectable in a number of tissues 3 days after injection.     

In Vitro Metabolism of T-2 Toxin

Incubation of T-2 toxin with the 9,000 x g supernatant fluid of both human and bovine liver homogenate resulted in conversion to a single, deacetylated product identified as HT-2 toxin. Metabolism is more rapid in human liver. HT-2 toxin was not produced when human plasma was the incubating medium nor was it produced by treatment of T-2 toxin with simulated gastric juice. T-2 toxin was stable in gastric juice for at least 1 h.     

The effects of four mycotoxins on the mitogen stimulated proliferation of bovine peripheral blood mononuclear cells in vitro

The effects of four mycotoxins, T-2 toxin, deoxynivalenol (DON), ochratoxin A (OTA) and fumonisin B1 (FB1) on the response of bovine peripheral blood mononuclear cells (PBM) in vitro to the mitogens concanavalin A (Con A), phytohaemagglutinin A (PHA) and pokeweed mitogen (PWM) were assayed after 4 days' incubation using 3H-thymidine uptake and the MTT bioassay. The concentrations of mycotoxin required to reduce the proliferative response of PBM by 50% for Con A, PHA and PWM as measured by 3H-thymidine incorporation was for T-2 toxin 0.30, 0.40 and 0.18 ng ml-1; for DON 0.07, 0.09 and 0.04 μg ml-1; for OTA 0.10, 0.20 and 0.15 μg ml- 1, and for FB1 35, 18 and 11 μg ml-1M by 50% for Con A, PHA and PWM as measured by the MTT bioassay were for T-2 toxin 2.0, 2.0 and 1.0 mg ml-1; for DON 0.70, 0.50 and 0.50 μg ml-1; OTA 1.5, 1,5 and 1.5 μg ml- 1; and FB1 >50, >50 and 20 μg ml-1 respectively. Further cytotoxicity assays including the LDH bioassay and Trypan blue exclusion were performed only on Con A-stimulated PBM cells after 72 h incubation. With the LDH-bioassay the 50% inhibition levels were T-2 toxin 0.3 ng ml-1, DON 0.4 μg ml- 1, OTA 1.4 μg ml-1 and FB1 3.5 μg ml-1; for Trypan blue uptake the 50% inhibition levels were T-2 toxin 5 ng ml-1, DON 2.3 μg ml-1 and OTA 4 μg ml-1 respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immunotoxicity of repeated low level exposure to T-2 toxin,a trichothecene mycotoxin,in CD-1 mice

Male CD-1 mice were gavaged with T-2 toxin (0.0–5.0 mg/kg body weight) every third day. Body weight gain was depressed by exposure to 2.5 mg/kg, or greater, T-2 toxin; this was not associated with decreased food intake. The weights of the liver, kidney, spleen, and thymus were affected by two weeks exposure to T-2 toxin. However, a persistent effect after four weeks was observed only for the thymus. Peripheral leucocyte counts were elevated in the highest dose groups after two and four weeks. Thymidine uptake by cells not simultaneously exposed to mitogen was increased in splenic cell cultures of mice exposed to 2.5 mg/kg T-2 toxin for two or four weeks. Phytohemagglutinin stimulation of splenic lymphocytes following two weeks of exposure was depressed in the 2.5 mg/kg dose group; this phenomenon was not observed after four weeks exposure. Response to pokeweed mitogen increased after four weeks of exposure to 2.5 mg/kg T-2 toxin. A delayed-type hypersensitivity response decreased following two weeks exposure to levels greater than 0.02 mg/kg. Production of I g M class antibodies by splenic lymphocytes, evaluated by a hemolytic plaque response to sheep erythrocytes, was depressed in the 2.5 mg/kg dose group after two weeks exposure to T-2 toxin. The sensitivity and specificity of T-2 toxin immunotoxicity was indicated by the various parameters evaluated.     

Characterisation of hemolysis induced by T-2 toxin

The erythrocyte constitutes a good model system for the study of membrane-associated toxicity events caused by the trichothecene mycotoxin, T-2. This study confirms that T-2 has a direct lytic effect on erythrocytes. Lysis of guinea pig red cells requires approx. 10(10) molecules/cell and reaches plateau values after 4-6 h. An activation energy, Ea approximately equal to 4.5 kcal was derived from the Arrhenius equation. By use of osmotic blockers of differing Stokes' radii, the functional size of the membrane lesion caused by T-2 toxin was shown to be smaller than 5.5 A. It is concluded that T-2 toxin may exert its toxic effects via the cell membrane.     

Intraperitoneal administration of carrier erythrocytes in dogs: an improved method for delivery of L-asparaginase

Carrier erythrocytes were prepared to encapsulate L-asparaginase by a hypotonic dialysis process. Dogs received either intravenous or intraperitoneal injections of cells containing both L-asparaginase and the marker [3H]inulin. The route of administration had no effect on the circulating survival of carrier erythrocytes. For enzyme therapy, intraperitoneal injection of a large volume of cells is technically feasible. Cells circulate with a maximum of 30% of the cells reaching circulation 24 h after injection. Those cells reaching circulation have a 7-day half-life for the encapsulated enzyme L-asparaginase.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

Functional alterations induced by fusarium T-2 toxin in Madin-Darby bovine Kidney (MDBK) and primary fetal bovine Kidney (PFBK) cultures

We recently reported that primary fetal bovine Kidney (PFBK) cells were consistently more sensitive to the cytotoxic effects of fusarium T-2 toxin than Madin-Darby bovine kidney (MDBK) cells in culture. The present report examined the influence of T-2 on selected biochemical parameters of these two culture types. T-2 toxin inhibited incorporation of labeled thymidine, uridine, and leucine in both culture types; at lower concentrations of the toxin, PFBK cells were affected to a greater extent than MDBK cells. T-2 toxin inhibited both the transport of thymidine as well as thymidine incorporation into macromolecules in MDBK cells during initial periods, but did not affect uridine incorporation. The cellular enzymes, K⁺- dependent phosphatase and succinic dehydrogenase were inhibited in MDBK but not in PFBK cultures; acid phosphatase was not influenced in either culture types. In a cell-free system none of the above enzymes were affected by T-2 until the toxin concentration exceeded 10^?5M.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp.

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Effect of toxins on arachidonic acid metabolism in rat cultured pulmonary alveolar macrophages

The action of a trichothecene (T-2), microcystin-LR and saxitoxin on arachidonic acid metabolism in cultured rat alveolar macrophages was studied. Pulmonary macrophages exposed to T-2 trichothecene were stimulated to synthesize and release large amount of thromboxane B2 (TxB2) and 6-Keto F1 alpha. Microcystin-LR induced significant release of prostaglandins F2 alpha (140%), PGE2 (175%) and TxB2 (169%) compared to controls. Saxitoxin induced TxB2 release by 37%. Arachidonic acid release was stimulated by all three toxins. The release of arachidonic acid and its metabolites in alveolar macrophages exposed to T-2 toxin was partially blocked by fluocinolone (1 microM). These results suggest that macrophages synthesize and release inflammatory mediators in response to toxin exposure, and fluocinolone may protect against T-2 toxicosis.     

Effect of T-2 toxin on feed intake, digestion and pathology of rabbits

Feed containing sublethal T-2 toxin concentrations (12.5 and 25 ppm) was fed to adult rabbits. The animals ate 60-70% less toxin-containing food. The dry matter content of their feces decreased significantly (on an average by 10%). The nutrient digestibility of the feed containing 12.5 ppm T-2 toxin, was increased by 2-6% and that of the 25 ppm T-2 toxin level decreased by 4-11% as compared to the control values. The rabbits showed emaciation, subacute catarrhal gastritis, necrosis of the lymphoid cells of the intestinal mucosa, depletion and necrosis in the lymphoid follicles of the ampulla ilei, spleen and lymph nodes. Necrosis of the cells of mononuclear phagocyte system and myeloid hemacytogenesis was characteristic. The toxin concentration of feces, cecotroph and urine was proportional to intake.     

Hepatic or splenic targeting of carrier erythrocytes: a murine model

Carrier mouse erythrocytes, i.e., red cells, subjected to a dialysis technique involving transient hypotonic hemolysis and isotonic resealing were treated in vitro in three different ways: (a) energy depletion by exposure for 90 min at 42 degrees C; (b) desialylation by incubation with neuroaminidase; and (c) oxidative stress by incubation with H2O2 and NaN3. Procedure (c) afforded maximal damage, as shown by analysis of biochemical properties of the treated erythrocytes. Reinfusion in mice of the variously manipulated erythrocytes following their 51Cr labeling showed extensive fragilization as indicated by rapid clearance of radioactivity from the circulation. Moreover, both the energy-depleted and the neuraminidase-treated erythrocytes showed a preferential liver uptake, reaching 50 and 75%, respectively, within 2 h. On the other hand, exposure of erythrocytes to the oxidant stress triggered a largely splenic removal, accounting for almost 40% of the reinjected cells within 4 h. Transmission electron microscopy of liver from mice receiving energy-depleted erythrocytes demonstrated remarkable erythrocyte congestion within the sinusoids, followed by hyperactivity of Kupffer cells and by subsequent thickening of the perisinusoidal Disse space. Concomitantly, levels of serum transaminase activities were moderately increased. Each of the three procedures of manipulation of carrier erythrocytes may prove applicable under conditions where selective targeting of erythrocyte-encapsulated chemicals and drugs to either the liver or the spleen has to be achieved.     

A yeast bioassay for trichothecenes

Like all eucaryotic cells, yeasts are sensitive to trichothecenes, especially T-2 toxin and verrucarin A. Based on this sensitivity, a yeast bioassay was developed to evaluate the toxicity of corn samples. The bioassay was optimized using spiked maize extracts. The toxicity of samples was defined as toxicity equivalent to a certain concentration of T-2 toxin standards. The assay can be performed on crude extracts, but the results are more precise after column clean-up. The test can also be used for the screening of trichothecene toxicity in general. The relative standard deviation (RSD) at 85 % growth inhibition (EC85) was 4.5% for the T-2 toxin standards (n = 8). This corresponds to an initial T-2 toxin concentration of approximately 58 ppb in the corn sample. Samples containing 188 and 113 ppb T-2 toxin caused a growth inhibition higher than 85%, whereas samples with toxin concentrations of 56 and 19 ppb had a growth inhibition less than 85%. Therefore the test can be used for the qualitative evaluation of corn samples up to a level of 58 ppb +/- 2.8 ppb. The bioassay is easy to perform with minimum requirements for equipment. Results can be obtained within 24 h and a large number of samples can be analysed daily. The costs are low and the results obtained are repeatable. With some modifications this test can be used for toxicity studies on trichothecene metabolites as well as for extracts with unknown compounds with properties similar to trichothecenes.     

Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting.

Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM-1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross-linking protein that facilitates anti-PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM-directed vascular immunotargeting of a reporter enzyme (beta-galactosidase, beta-Gal) in intact animals. Intravenous injection of [125I]SA-beta-Gal conjugated with either anti-PECAM or IgG led to a high 125I uptake in liver and spleen, yet beta-Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti-PECAM/[125I]SA-beta-Gal, but not IgG/[125I]SA-beta-Gal, accumulated in the lungs (36.0+/-1.3 vs. 3.9+/-0.6% injected dose/g) and induced a marked elevation of beta-Gal activity in the lung tissue persisting for up to 8 h after injection (10-fold elevation 4 h postinjection). Using histochemical detection, the beta-Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti-PECAM carrier also provided 125I uptake and beta-Gal activity in the renal glomeruli. Predominant intracellular localization of anti-PECAM/SA-beta-Gal was documented in the PECAM-expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell-selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.     

Receptor-mediated gene delivery and expression in vivo

A soluble DNA carrier system was used to target a foreign gene specifically to liver in vivo via asialoglycoprotein receptors. The DNA carrier was prepared consisting of a galactose-terminal (asialo-)glycoprotein, asialoorosomucoid (AsOR), covalently linked to poly-L-lysine. The conjugate was complexed in a 2:1 molar ratio (based on AsOR content of the conjugate) to the plasmid, pSV2 CAT, containing the gene for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Intravenous injection of [32P]plasmid DNA complexed to the carrier demonstrated specific hepatic targeting with 85% of the injected counts taken up by the liver in 10 min compared to only 17% of the counts when the same amount of [32P]DNA alone was injected under identical conditions. Targeted pSV2 CAT DNA was detected at a level of 1.0 ng/g liver by hybridization of a [32P]pSV2 CAT cDNA probe to rat liver DNA extracted 24 h after intravenous injection of AsOR-poly-L-lysine-DNA complex containing 1.0 mg of DNA. Homogenates of livers taken 24 h after injection of the complex revealed that the targeted CAT gene was functional as reflected by the detection of CAT activity (approximately 4 microunits/mg protein). Livers from control animals that received individual constituents of the complex produced no CAT activity. Simultaneous injection of excess AsOR to compete with the AsOR-poly-L-lysine-DNA complex for uptake by the liver inhibited CAT gene expression. Assays for CAT activity in other organs (spleen, kidney, lungs) failed to demonstrate any activity in these organs. This new soluble DNA carrier system can permit targeted delivery of foreign genes specifically to liver with resultant foreign gene expression in vivo.     

Impact of flow rate on lactate uptake and gluconeogenesis in glucagon-stimulated perfused livers

The impact of reduced hepatic flow on lactate uptake and gluconeogenesis was examined in isolated glucagon-stimulated perfused livers from 24-h-fasted rats. After surgical isolation, livers were perfused (single pass) for 30 min with Krebs-Henseleit (KH) bicarbonate buffer, fresh bovine erythrocytes (hematocrit approximately 20%), and no added substrate. After this "washout" period, steady-state perfusions were initiated with a second reservoir containing the KH buffer, bovine erythrocytes, [U-(14)C]lactate (10,000 dpm/ml), lactate (2.5 mM), and glucagon (250 microg/ml). Perfusion flow rate was adjusted to one of five rates (i.e., 1.8, 2.7, 3.9, 7.4, and 11.0 ml.min(-1).100 g body wt(-1)). After the perfusion, the liver was dissected out and weighed so as to establish the actual flow rate per gram of liver. The resulting flow rates ranged from 0.52 to 4.03 ml.min(-1).g liver(-1). As a function of flow rate, lactate uptake rose in a hyperbolic fashion to an apparent plateau of 2.34 micromol.min(-1).g liver(-1). Fractional extraction (FX) of lactate from the perfusate demonstrated an exponential decline with increased flow rates (r=0.97). At flow rates above 1.0 ml.min(-1).g liver(-1), adjustments in FX compensated for changes in lactate delivery, resulting in steady rates of lactate uptake and gluconeogenesis. Below 1.0.min(-1).g liver(-1) the increased FX was unable to compensate for the decline in lactate delivery and lactate uptake declined rapidly. Gluconeogenesis demonstrated similar kinetics to lactate uptake, reflecting its dominant role among pathways for lactate removal under the current conditions.