Dose Of Ricin Research Articles

Intrapulmonary Delivery of Ricin at High Dosage Triggers a Systemic Inflammatory Response and Glomerular Damage

In view of the possibility that ricin may be used as a bioweapon against human populations, we examined the pathological consequences that occur in mice after introduction of ricin into the pulmonary system. Intratracheal instillation of a lethal dose of ricin (20 microg/100 g body weight) resulted in a hemorrhagic inflammatory response in multiple organs, accompanied by activation of mitogen-activated protein kinases, increased synthesis of proinflammatory RNA transcripts, and increased levels of circulating cytokines and chemokines. A sublethal dose of instilled ricin (2 microg/100 g body weight) induced a similar response in lungs but did not cause detectable damage in other organs. Lungs of mice that recovered from a sublethal dose of ricin displayed evidence of fibrosis and residual damage. A lethal dose of ricin caused accumulation of proinflammatory RNA transcripts and substantial damage to 28S rRNA of multiple organs, including lung, kidney, spleen, liver, and blood, demonstrating that instilled ricin gained access to the circulation. The kidneys of mice instilled with a lethal dose of ricin showed accumulation of fibrin/fibrinogen in glomerular capillaries, increased numbers of glomerular leukocytes, and impairment of kidney function. A sublethal dose of ricin failed to induce damage to 28S rRNA in kidney or other extrapulmonary organs.

Fatal Ricin Toxicosis in a Puppy Confirmed by Liquid Chromatography/Mass Spectrometry when Using Ricinine as a Marker

Ricin, a lectin from the castor bean plant (Ricinis communis), is considered one of the most potent plant toxins. Ingestion of masticated seeds results in high morbidity, with vomiting and watery to hemorrhagic diarrhea. The prognosis varies with the number of seeds ingested, the degree of mastication, individual susceptibility, and the delay in treatment. Low mortality restricts assessment of histologic lesions, and the literature on toxicologic analysis for ricin is limited. This report describes a fatal case of castor bean ingestion in a 12-week-old Mastiff puppy, with confirmation of ricin exposure through detection of the biomarker ricinine by liquid chromatography/mass spectrometry (LC/MS). Despite supportive therapy, the puppy died several hours after presentation for acute vomiting, diarrhea, and lethargy. At necropsy, a segment of jejunum and mesenteric lymph nodes were congested. When the owner reported the presence of castor beans in the dog's feces, selected formalin-fixed and unfixed tissues were submitted for diagnostic evaluation. Histopathologic findings included superficial necrotizing enteritis of the jejunum and occasional, random foci of coagulative necrosis in the liver. The alkaloid ricinine was detected in gastric content by using a newly developed LC/MS method. This confirmation of exposure is important in the diagnosis of ricin toxicosis, because ingestion of castor beans is not always fatal, histologic lesions are nonspecific, and the degree of mastication can influence the effective dose of ricin.

Pulmonary gene expression profiling of inhaled ricin

Aerosol exposure to ricin causes irreversible pathological changes of the respiratory tract resulting in epithelial necrosis, pulmonary edema and ultimately death. The pulmonary genomic profile of BALB/c mice inhalationally exposed to a lethal dose of ricin was examined using cDNA arrays. The expression profile of 1178 mRNA species was determined for ricin-exposed lung tissue, in which 34 genes had statistically significant changes in gene expression. Transcripts identified by the assay included those that facilitate tissue healing (early growth response gene (egr)-1), regulate inflammation (interleukin (IL)-6, tristetraproline (ttp)), cell growth (c-myc, cytokine-inducible SH2-containing protein (cish)- 3), apoptosis (T-cell death associated protein (tdag)51, pim-1) and DNA repair (ephrin type A receptor 2 (ephA2)). Manipulation of these gene products may provide a means of limiting the severe lung damage occurring at the cellular level. Transcriptional activation of egr-1, cish-3, c-myc and thrombospondin (tsp)-1 was already apparent when pathological and physiological changes were observed in the lungs at 12 h postexposure. These genes may well serve as markers for ricin-induced pulmonary toxicity. Ongoing studies are evaluating this aspect of the array data and the potential of several genes for clinical intervention.

Morphology of ricin and abrin exposed endothelial cells is consistent with apoptotic cell death.

Cultures of bovine pulmonary endothelial (BPE) cells were exposed to LC70 doses of ricin or abrin (15.5 and 4.5 pM respectively) over a period of up to 40 h. The viability of the cultures (as determined by the neutral red (NR) dye retention assay) declined after 6 h exposure to the toxins. From 15 h onwards, cellular material in toxin exposed cultures became detached from the substratum of the culture vessels. Hoffman modulation contrast photomicrography showed that this process was due to ricin and abrin exposed cells collapsing into membrane bound vesicles which retained the NR dye, became detached and floated into the medium. These apoptotic-like structural changes were further investigated by transmission electron microscopy (TEM) and by agarose gel electrophoresis of DNA from control and exposed cultures. Many of the characteristic changes associated with apoptotic cell death were seen using TEM, including heterochromatin condensation at the nuclear periphery, crenulation of the nuclear membrane and progressive degeneration of residual nuclear and cytoplasmic structures. The plasma membrane of many cells remained intact, and contained nuclear and cytoplasmic debris. Agarose gel electrophoresis of DNA extracted from toxin-treated cells revealed oligonucleosome sized DNA fragments, characteristic of apoptosis, from adherent cells at 7 h and both adherent and floating populations when harvested from 15 h; DNA from unexposed control cells did not show this fragmentation. The identification of apoptosis as being a significant additional mechanism of toxicity following exposure to ricin and abrin holotoxins raises the possibility of developing new therapeutic strategies against poisoning by these phytotoxins.

Drugs that show protective effects from ricin toxicity in in vitro protein synthesis assays.

We used an in-vitro, inhibition of protein synthesis assay (PSI) to test a wide variety of drugs for possible therapeutic use against ricin, a toxic glycoprotein that causes death in animals by inhibiting protein synthesis. Selection of test drugs was based on possible interference with ricin activity at different stages of the toxic process. Most of the drugs tested had no effect on ricin-induced PSI, were toxic when tested alone, or enhanced the toxicity of ricin. The only ones showing protection were galactose, lactose, and several derivatives of these sugars, Brefeldin A (BFA), 3'-azido-3'-deoxythymidine (AZT), and a purine derivative (BM33203). THe sugar derivatives provided 50% protection against a PSI ED99 of ricin (0.1 micrograms/ml). Concentrations of BFA greater than 0.5 micro M caused about 50% PSI by itself, but blocked any further inhibitory effects of ricin. AZT, at optimum concentrations, reached a maximum protection level of about 40% in the presence of an ED99 dose of ricin, while the nucleoside derivative, BM33203 and AZT appeared to have an additive effect, showing up to 80% protection from an ED99 dose of ricin. Drugs showing protection in the PSI cell assay showed no protection from ricin in a cell-free translation assay used to determine if they would block ricin at the protein synthesis site.

Effects of ricin on the ability of rabbit arteries to contract and relax.

Ricin, a toxic lectin from castor beans, reduces blood pressure. The current studies determined the effect of ricin on contractions in response to norepinephrine (NE) of rabbit central ear artery, endothelium-dependent relaxations to methacholine and ATP of aorta rings and endothelium-independent relaxations to papaverine in central ear artery and aorta rings. Rabbits were given 0.11 or 0.22 micrograms kg-1 ricin i.v. and 18 h, 4 days, or 7 days later the arteries were removed and tested. Maximal contractions to NE were increased by 8-23% (P > 0.05) with ricin treatment. The EC50 was increased in five of six ricin-treated groups, although only significantly so at 18 h after a minimum lethal dose of ricin. Maximum relaxations to methacholine were increased by 20-57% (P > 0.05) at 18 h following both ricin doses but returned to control values at later time points. Relaxations in response to ATP were significantly enhanced in all ricin-treated groups (95-205%) except in the 18 h and 4-day 0.11 microgram kg-1 dose groups where the increase was 0-46%. Relaxations in response to papaverine were not altered in either artery. Thus, ricin decreases the sensitivity of the rabbit central ear artery to NE and increases endothelial-dependent relaxations of the rabbit aorta. Therefore, decreased blood pressure following ricin administration may be due to vasodilation caused by decreased vascular contractions and increased endothelial-dependent vascular relaxation.

The effects of ricin on the sympathetic vascular neuroeffector system of the rabbit.

Ricin is a toxic lectin that inhibits protein synthesis. Because ricin decreases arterial pressure and causes cardiovascular collapse, its effects on the vascular neuroeffector system were investigated. Rabbits were given either of two doses of ricin, and then norepinephrine (NE) release from aorta to transmural stimulation, NE uptake into aorta, NE content of aorta, monoamine oxidase activity, and catechol-O-methyl transferase activity in aorta were determined 18 hours, 4 days or 7 days later. Norepinephrine uptake and enzyme activities in the aorta were not altered by ricin administration. Norepinephrine release and content of aorta were increased at most time periods following ricin administration, significantly so for NE content at 4 days and for release at 18 hours following the lower dose of ricin. We conclude that the mechanisms involved in the release of NE from sympathetic nerves in the vasculature are not impaired by ricin administration, but rather show changes that indicate increased compensatory activity.

Modulation of ricin toxicity in mice by biologically active substances.

Ricin is a highly toxic protein produced by the castor bean (Ricinus communis). It is one of various protein toxins that consist of two subunits joined by a disulfide bridge. One chain facilitates entry of the toxin into the cell while the other chain exhibits RNA N-glycosidase activity, which attacks a specific site on 28S rRNA, preventing polypeptide elongation and leading to cell death. Although ricin and other protein toxins are potential health hazards, no antidote against these toxins exists. Thus, a number of selected compounds were screened for their ability to alter ricin lethality in mice, based on percentage survival and time to death following a ricin LD100 of 25 micrograms kg-1 i.p. While no compound tested prevented lethality, dexamethasone and difluoromethylornithine (DFMO) significantly extended survival time. The effects of DFMO on ricin toxicity were markedly influenced by altering various pharmacokinetic parameters. The antioxidants butylated hydroxyanisole and vitamin E succinate also extended survival time in response to a lethal dose of ricin, but to a lesser extent than did dexamethasone and DFMO. The Golgi apparatus inhibitors monensin, swainsonine and tunicamycin enhanced ricin toxicity, as evidenced by shortened survival times. In addition, various nucleoside analogs, including acyclovir and trifluridine as well as adenosine, guanosine and dibutyryl cyclic AMP, also potentiated the toxicity of ricin. The results demonstrate that the toxicity of ricin is modulated by a wide variety of structurally distinct chemicals and may involve different mechanisms. Furthermore, the extent and direction of the modulation of ricin toxicity is highly dependent upon pharmacokinetic variables, including dose and dosing interval.

Protection of mice from inhaled ricin by vaccination with ricin or by passive treatment with heterologous antibody

Mice were vaccinated subcutaneously with 25 μg kg −1 of ricin in the presence of Freund's complete adjuvant or Ribi™ adjuvant, followed by a boost 14 days later with 50 μg kg −1 ricin in Freund's incomplete adjuvant or Ribi adjuvant, respectively. Three subsequent boosts at 28-day intervals with 25 μg kg −1 ricin yielded high anti-ricin antibody titres as determined by ELISA. Vaccinated animals were exposed to an aerosolized LD 99 dose of ricin. With the exception of one death not attributable to ricin intoxication, all vaccinated mice survived the lethal aerosol exposure. In addition, a passive protection regimen was evaluated in mice pretreated with 100 μg purified goat anti-ricin IgG administered intravenously, and then challenged with ricin intravenously. All were resistant to 125 μg kg −1 of ricin, a dose greater than 25 times the intravenous lethal dose. Mice injected intravenously with 5 mg of the same IgG were protected from a lethal aerosol challenge. These results indicated that it is possible to protect animals from inhaled ricin by vaccination or passive administration of specific antibodies.

In vivo potentiation of ricin toxicity by monensin delivered through liposomes

Monensin, a carboxylic ionophore which is known to raise intravesicular pH, was intercalated in liposomes and its effect on the toxicity of ricin in mice was studied. The toxicity of ricin in vivo was found to be significantly enhanced by the administration of monensin intercalated in liposomes (liposomal monensin). The observed enhancement of the toxicity of ricin by monensin was highly dose-dependent and was maximal when ricin was injected within 60 min of monensin injection. The survival time was found to be reduced in the range of 8–20 h, depending on the dose of ricin used, by liposomal monensin. Stability of liposomes containing monensin as inferred from the release of entrapped calcein or FITC-dextran under both in vivo and in vitro conditions was comparable to that observed for liposomes without monensin. Liposomal monensin remains in circulation for 2 h and was cleared from the blood stream after 4 h. In contrast, 15 min was required for the clearance of monensin when administered in free form. Studies on the distribution of liposomal monensin and 125I-ricin in various tissues have revealed that monensin is mainly localized in the liver and spleen which are also the major sites for ricin accumulation. Our observation on the substantial enhancement of ricin toxicity in vivo by liposomal monensin strongly supports the potential usefulness of the latter as a potentiating agent in the enhancement of the toxicity of immunotoxin or hormonotoxin for selective elimination of cancer cells.

Dose- and time-dependent selective and non-selective effects of ricin (RCA 120) on rat primary sensory neurons

The distribution of degenerating fibers in the spinal cord was studied in Fink-Heimer-stained sections following treatment of tibial nerve with ricinus communis agglutinin (RCA 120). The ricin was either injected into the nerve or applied in a capsule on the transected nerve. Short survival times and low doses of ricin resulted in degeneration in somatotopically appropriate parts of the medial dorsal horn. Longer survival times and higher doses resulted in degeneration which progressively expanded into inappropriate areas in the central and lateral parts of the dorsal horn and in deeper laminae regardless of the mode of application. Furthermore, the effect of a ricin injection into the tibial nerve on transganglionic transport of choleragenoid horseradish peroxidase (B-HRP) in the peroneal nerve was studied following a simultaneous or delayed B-HRP injection. A simultaneous ricin and a B-HRP injection resulted in primary afferent HRP labeling in the gray matter, regardless of the dose of ricin. Following a delayed B-HRP injection almost no primary afferent labeling was seen in the gray matter, unless a very low dose of ricin was injected. This study shows that treatment of a peripheral nerve with a high dose of ricin and a long survival time may result in a considerable non-selective degeneration of fibers in the spinal cord. A selective degeneration may, however, be obtained by using lower doses or shorter survival times.

Experimental treatment of neuromas in the rat by retrograde axoplasmic transport of ricin with selective destruction of ganglion cells

In this experiment the toxic lectin, ricin, was injected into the terminal proximal segment of transected rat sciatic nerves to evaluate its effect on neuroma formation. The dosage of ricin injected ranged from 0.5 μg to 1.5 μg. All animals survived and all treated nerves showed no evidence of neuroma formation and no evidence of viable nerve elements 1 month after treatment. The contralateral untreated nerves in the experimental groups, as well as those of the control group, all showed neuroma formation. This study demonstrates the effectiveness of small doses of ricin in preventing neuroma formation in rats. The mechanism of action of ricin, destruction of the parent ganglion cells of the treated peripheral nerves after its retrograde axoplasmic transport, is also demonstrated.

Human immune response to immunotoxins.

The potential value of immunotoxins (ITs) in the treatment of human cancer may eventually prove to be limited by the development of host antibodies against the conjugate. The development of antimurine antibodies after administration of unconjugated monoclonal antibodies is well documented and has been associated with more rapid murine antibody clearance and decreased clinical effect [1, 2]. Similarly, antitoxin antibodies have been detected in both animals and humans treated with ricin and abrin [3, 4]. When given lethal doses of ricin, survival was prolonged in mice which had developed antiricin antibody levels in excess of 10–20 ng/ml [3].

The toxic plant proteins ricin and abrin induce apoptotic changes in mammalian lymphoid tissues and intestine.

The toxins ricin and abrin are potent inhibitors of protein synthesis. Apoptosis has been shown to be induced in some cells by cycloheximide and actinomycin D whereas the process is prevented in other cells by the same agents, both inhibitors of protein synthesis. We were interested to find whether ricin and abrin caused any apoptotic changes in rapidly dividing tissues where we believed that these toxins concentrate. Rats were injected intramuscularly with toxin and killed at time intervals, tissues being removed and examined by light and electron microscopy. Apoptotic-like bodies were abundant in para-aortic lymph nodes, Peyer's patches and ideal crypts of ricin or abrin intoxicated rats. Abrin was found to cause markedly more pronounced changes in these tissues, when compared with a similar dose of ricin. Prior to this, these toxins have been reported as causing necrosis in animal tissues.

560 EFFECT OF RICINUS COMMUNIS TOXIN (RICIN) ON RABBIT INTESTINAL EPITHELIUM: A MODEL OF PROTEIN-INDUCED MUCOSAL DAMAGE

Ricin inhibits protein synthesis in eukaryotic cells following binding to galactose membrane receptors and cytoplasmic internalization of its A subunit. Similar but less well defined mechanisms are probably involved in small bowel toxicity by certain food and bacterial proteins. We have therefore used ricin to develop an in vitro model of protein-induced small bowel epithelial damage using organ culture methodology. Rabbit jejunal mucosal explants were exposed (30 min, 25°) to varying doses of ricin (0.1-50 μg) and then cultured in ricin-free medium for 2 to 24 hours at 37°. Inhibition of protein synthesis (14C-leucine incorporation) occurred as a function of ricin dose and culture time. A t50 plot (1/ricin/t50) revealed a lag time of about 30 minutes for infinite ricin dose. Light and electron microscopy showed a progression of damage in epithelium which correlated to ricin dose and level of inhibition of protein synthesis. The effect of ricin was blocked by lactose and lactulose (100 mM) added during the ricin exposure period. The dose and time relationships of the effect of ricin on intestinal epithelium in culture suggest membrane responses and down regulation processes identical to those of endocytotic cells. This approach is therefore useful for investigations of the membrane and cellular mechanisms associated with protein-induced enterocyte toxicity.

Toxicity of abrin and ricin in mice and dogs.

Mice and dogs, were treated iv with the cytostatic proteins abrin and ricin and observed for clinical, biochemical, and morphological aberrations. In both mice and dogs death occurred within a narrow dose range. Dogs given toxic doses of ricin and abrin showed weakness, anorexia, apathy, and moderate fever. No signs attributable to the central nervous system were observed. Dogs dying from intoxication expired after 15-40 h. After nonlethal doses the animals recovered, apparently completely, in 1-3 wk. No delayed changed were observed in dogs after 4 mo. Abrin and ricin, in contrast to most other cytostatic agents, did not inhibit myelopolesis. However, after sublethal dpses a rapid but translent decrease of peripheral thrombocytes was observed. No evidence for specific liver damage or impairment of kidney function was obtained. Few abnormalities were observed at autopsy or on microscopic and electron microscopic examination of the tissues, in contrast to the findings of some earlier investigators. The results indicate that in mice and dogs given sublethal doses of highly purified toxins the symptoms are reversible. There was no finding militating against a phase 1 clinical trial.

Effect of ricin and abrin on survival of L1210 leukemic mice and on leukemic and normal bone-marrow cells.

The effect of ricin and abrin on the survival of mice treated with L1210 leukemic cells intraperitoneally or intravenously was studied. In mice given 1 X 10(5) L1210 leukemia cells intraperitoneally a single dose of ricin (2.1 microgram/kg) intraperitoneally gave the best results, an increased life span (ILS) of 59%. Abrin also increased the life span of such animals although to a lesser extent. The effect of ricin was superior to that of 5-fluorouracil, but inferior to that of adriamycin, which gave a maximum ILS of 280%. In mice given L1210 cells intravenously no increase in life span was obtained with ricin, abrin or adiramycin, whereas 5-fluorouracil gave an ILS of 40-50%. In spleen colony assays the differential effect of ricin and abrin on the proliferative capacity of normal hematopoietic and leukemic colony-forming cells in bone marrow was studied. The differential effect of ricin was as good as that of adriamycin and considerably better than that of 5-fluorouracil. Abrin had a much smaller effect than ricin on both normal and leukemic cells. The effect of abrin on the leukemic cells was too small to be of therapeutic value. The results warrant exploration of the use of ricin in the treatment of human leukemia.

Toxic effects of ricin: studies on the pathogenesis of liver lesions.

Rats treated with ricin, an inhibitor of protein synthesis, at the dose of 1 mug/100 g body weight, developed within 72 hours a severe liver necrosis. Protein synthesis was practically unchanged. The ultrastructural changes that ricin induces in rat liver were investigated. The earliest changes were observed in sinusoidal cells which were progressively damaged until they became necrotic. Only after the development of these lesions hepatocytes appeared to be damaged. The hypothesis is formulated that hepatocyte necrosis is a consequence of the disappearance of sinusoidal cells. This might explain why protein synthesis was unaffected in liver: since ricin exerted its toxic effect on the sinusoidal cells the inhibition was not detectable, these cells being "diluted" by the mass of parenchymal cells. Ricin at the dose of 10 mug/100 g body weight did not affect protein synthesis in the liver, but exerted a marked and precocious inhibition of protein synthesis in the spleen, which is very rich in reticuloendothelial cells. Moreover, a severe necrosis of the red pulp of the spleen was observed in rats poisoned with this dose of ricin.

Effect of Tris(Beta-Chloroethyl)Amine on Antibody-Production in Goats

Summary The serum antibody-content of goats which had been actively immunized against ricin was allowed to decline by cessation of antigen administration. When serum antibody had reached negative levels of activity, the injection of a single stimulating dose of ricin caused a prompt increase in circulating antibodies such that within one week levels were obtained equal to or greater than previous maxima. However, if immune goats with negative antibody-levels were injected with chronic, leucopenic doses of the nitrogen mustard, tris(beta-chloroethyl)amine, before the administration of the stimulating dose of ricin, anamnestic responses were delayed in all animals and in a few actually decreased by comparison with the responses of controls. The fact that prior treatment with amine mustard influenced the course of anamnesis was discussed in relation to the untoward effect of the treatment on leucopoietic tissues. It was considered reasonable to attribute untoward change in anamnestic response to a direct toxic action of the agent on leucopoietic tissues which was not mediated by primary stimulation of the adrenal cortex.

SPECIFIC ANTISERUM AGAINST RICIN

To the Editor:—On pages 485 and 486 of the September 1942 issue of theAmerican Journal of Diseases of Children, under the heading Case Reports, there is an article by Koch and Kaplan entitled Castor Bean Poisoning. In the last paragraph the authors state that there is no specific antidote for this condition. They further state that the mortality in a series of 150 cases was 6 per cent. As it is perfectly feasible to make an antiserum against the toxin, ricin, by inoculating rabbits with increasing doses of ricin, should not steps be taken by some society to induce one of the companies manufacturing biologicals to prepare this specific antiserum and have the fact that this exists publicized, so that it may be used in future cases of castor bean poisoning? It would seem logical that the mortality rate could thus be reduced to a minimum. I suppose