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Ricin is a protein extracted from the seeds of the castor bean plant (Ricinus communis). Ricin is a lectin and a member of a group of ribosome-inactivating proteins like abrin (from the seeds of the rosary pea, Abrus precatorius), that prevent synthesis of protein in eukaryotic ribosomes. Ricin is one of the most toxic substances and as such was once known to be a chemical weapon (Agent W) but is now subject to the Chemical Weapons Convention. However, ricin has been used as a poison for criminal and terrorist purposes.
Ricin is an extremely toxic poison, and thus can kill even if applied in a small amount. Thus, the introduction of rapid, highly sensitive chemical and biological/immunological analytical methods capable of detecting the toxin at or below ng/ml level is of vital importance (see, for example, Kalb and Barr, 2009; Lubelli et al., 2006; Uzawa et al., 2008). In cases where castor beans or crude ricin preparations are the possibly dangerous poisons, ricinine can also serve as a biomarker (see, for example, Mouser et al., 2007).
The chemical is particularly deadly because it can be inhaled, ingested, or swallowed and is quickly broken down in the body and is virtually undetectable. There is currently no antidote to ricin, although a prospective vaccine has been developed that has been successfully tested in mice. Ricin is a potent protein cytotoxin derived from the beans of the castor plant. Castor beans are omnipresent worldwide, and the toxin is fairly easy to extract; therefore, ricin is widely available. Ricin's significance as a potential BW [biological warfare] toxin relates in part to its wide availability.
HOW IT IS OBTAINED
Ricin toxin, found in the bean of the castor plant, Ricinis communis, is one of the most toxic and easily produced plant toxins. The shrub like ornamental castor plant, Ricinus communis (Euphorbiaceae), originated in Africa and Asia, and has been cultivated and distributed throughout the world. The annual production of castor oil seed exceeds over 1 million metric tonnes (Food and Agricultural Organization of the United Nations, 2009). Ricin is a byproduct of castor oil production: when castor beans are crushed, they form a pulp from which castor oil is extracted, and ricin is what remains. The waste mash from this process is 3-5% ricin by weight. The seeds of the plant have three main constituents: oil, castor oil, which is the glyceride of ricinoleic acid; a mildly toxic alkaloid ricinine and several isoforms of a highly poisonous glycoprotein ricin present up to 5% in the seeds.
Although adaptable to a wide temperature range, it fails to resist to subfreezing temperatures and withstand best in elevated year-round temperatures. Brazil, Ecuador, Ethiopia, Haiti, India, and Thailand are the countries which commercially cultivate most of the seeds. Castor oil is found in many commonly used substances such as paints, varnishes, and lubricating oils, and is also used as a purgative. After oil isolation, the remaining seed cake may be detoxified by heat treatment and used as an animal feed supplement. The seed hulls are similar to barnyard manure in their fertilizer value. The toxicity of castor beans has been known since ancient times and more than 750 cases of intoxication in humans have been described. Although ricin's lethal toxicity is approximately 1,000-fold less than that of botulinum toxin, ricin may have significance as a biological weapon because of its heat stability and worldwide availability, in massive quantities, as a by-product of castor oil production.
B. DESCRIPTION OF THE AGENT
Identity and Physicochemical Properties
Ricin is a 66-kilodalton (kd) globular protein that makes up 1% to 5% by weight of the bean of the castor plant, Ricinis communis. The CAS Registry Number of ricin is [9009-86-3]. In a pure state, ricin is a white crystalline powder. It is a water-soluble glycoprotein consisting of two polypeptides, termed A and B chains, which are linked by a disulfide bond (ASSB). The amino acid sequence of ricin (or ricin D as the toxic fraction from the beans is called) was resolute by Funatsu et al. (1978, 1979).
The A chain contains 265 amino acids and has a molecular weight of 32 kDa; its sugar content is 2.6%. The isoelectric point of the A chain is 7.34. The B chain of 260 amino acids and four internal disulfide bonds has a molecular weight of 34 kDa and its sugar content is 6.4%. The A chain has enzymatic properties (ribosomal RNA N-glycosidase, EC 220.127.116.11) responsible for the toxicity of ricin, while the B chain is a lectin binding to galactose-containing glycoproteins and glycolipids on the surface of target cell components. The use of X-ray crystallography studies was used to solve the three-dimensional structure of ricin (reviewed by Lord et al., 1994). The physicochemical and photochemical properties of ricin have recently been assessed (Gaigalas et al., 2007).
Because the chaff left over from castor oil processing can be used to feed cattle, a great deal of effort was dedicated to its detoxification (Balint, 1974; European Food Safety Authority, 2008). High-temperature denaturing (>80Â°C for 1 h) and chemical methods (oxidation with potassium permanganate, hydrogen peroxide, iodine, etc.) were put forward to destroy the toxin (see, for example, Barnes et al., 2009). In the presence of 2-mercaptoethanol, which reduces the disulfide bond joining the A and B chains, the toxicity of ricin is lost; removal of 2-mercaptoethanol, however, allows the reconstitution and reactivation of the toxin. Ricin is degraded by papain but only slowly by trypsin. The fate of ricin in the body is incompletely understood.
The toxin-rich chaff byproduct of castor oil manufacture has been used to kill mice and moles. Conjugates of ricin and cell-specific antibodies are experimental anticancer immunochemotherapeutic agents (Sandvig and van Deurs, 2005; Stirpe et al., 1992). Recently, transgenic rice and maize engineered to produce a fusion protein comprising the Cry1Ac endotoxin of Bt and the ricin B lectin subunit have proved to be insecticidal to insects that are otherwise able to endure Cry toxins (Mehlo et al., 2005).
Mode of action
Once it was thought that the toxic action of ricin preparations in mammals is due to its hemagglutinating effect, but this activity was shown to be associated with the structurally similar, but nontoxic agglutinins present in the castor bean. It is now well established that ricin inhibits protein synthesis in eukaryotic systems by catalytically inactivating the 60S subunit involved in the translation process. The structural aspects of biochemical interaction of ricin and other ribosome-inactivating protein (RIP) toxins from plants and fungi were entirely assessed (Kozlov et al., 2006; Stirpe and Battelli, 2006).
Briefly, the B chain binds to galactose/N-acetylgalactosamine-containing glycoproteins and glycolipids in eukaryotic cells. The binding to surface receptors can be inhibited by galactose or lactose in vitro. It appears that both chains facilitate the penetration by endocytosis of the toxin into the cell. The B chain, however, aids the toxin in translocating to endosomal targets as well. Once in the cytosol, the A chain cleaves a single adenine base from the 28S ribosomal (Ribonucleic acid) RNA within the 60S ribosomal subunit, rendering it unable to bind the elongation factor 2 which consequently leads to an arrest of protein synthesis. A single A chain molecule can inactivate 1500 ribosomes per minute and kill the cell. In addition to inhibiting protein synthesis, ricin was shown to provoke apoptosis, cause oxidative stress, release proinflammatory cytokines, modify cell membrane structure and function, and impair nuclear DNA (reviewed by Stirpe and Battelli, 2006). Lipase activity of ricin was also clearly shown (Morlon-Guyot et al., 2003).
Toxicity to Laboratory Animals
Although the mode of action of ricin at the molecular level is known, the mechanisms responsible for the clinical and lethal effects of the toxin are still inadequately understood. Representative animal toxicity data for ricin administered by different routes are shown in Table 1. The variations in the acute toxicity values reported in the literature are mainly due to mixture of the preparations used in the tests (reviewed by Balint, 1974). The symptoms of ricin poisoning manifest slowly, usually 12 hr after administration, and include rather sudden outbursts of convulsions and opisthotonos, followed by paralysis of the respiratory center, eventually leading to death. Fortuitous poisoning is usually due to ingestion of castor beans (for recent examples, see Aslani et al., 2007; Soto-Blanco et al., 2002). Laboratory tests with seeds showed hen to be the most resistant species (the lethal dose was 14 g/kg); sheep and horse were more sensitive (lethal doses were 1.25 and 0.10 g/kg, respectively). The toxin is pyrogenic in mammals (Balint, 1993). In the serum of animals treated with ricin, antibodies specific to ricin have repeatedly been detected (see, for example, Griffiths et al., 2007).
Ricin is highly toxic upon injection and inhalation. Using transmission electron microscopy, Brown and White (1997) (see also Griffiths et al., 1995b, 2007) examined the histopathological changes in the lungs of rats upon ricin inhalation. The animals were exposed to an LCt30 (the concentration in air that killed 30% of the exposed animals) of 11.21 mg/min/m3 dose of the toxin. Necrotic changes were
evident in the capillary endothelium and type I epithelial cells, accompanied by intraalveolar edema 12-15 h after exposure.
Table 1. Acute Toxicity of Ricin
Minimal lethal dose (Î¼g/kg)
Mouse, various strains
a Ricin from R. communis var. "Hale Queen".
bRicin from R. communis var. zanzibarensis.
cRicin reconstituted from A and B chains.
Wong et al. (2007) have recently shown that tracheal instillation of sublethal dosages of ricin in mice causes localized inflammation in the lungs with minimal evidence of systemic effects, which is in accordance with earlier distribution studies. A lethal dose of the toxin, however, induces not only severe hemorrhagic inflammatory response in the pulmonary system but enters the vascular system and initiates inflammation in multiple organ sites, especially in the kidneys.
Wilhelmsen and Pitt (1996) examined in rhesus monkeys the inhalational toxicity of 21-41.8 Î¼g/kg doses of aerosolized ricin. The major intoxication symptom was a rapid onset of dyspnea occurring after a 20-24 hr lag period after exposure. Doses above 36.5 Î¼g/kg caused death 36-48 hr after exposure. In addition to skin elasticity indicating dehydration, autopsy findings confined to the exposed organ, that is the respiratory tract, thus differed from those reported for parenteral ricin intoxication in other species. They included clear foamy fluid in the trachea and mainstream bronchi; serous fluid in the thoracic cavity; diffusely wet, mottled red lungs with multifocal necrosis; acute inflammation of airways, and lesions along the pulmonary lymphatic drainage course. In some monkeys bilateral adrenocortical necrosis was also seen. The cause of death was attributed to inability to breathe following massive pulmonary alveolar flooding.
Toxicity to Humans
As with animals, the toxic effects of ricin have a delayed period of several hours after administration. The initial clinical symptoms of ricin poisoning are nonspecific and include general malaise, nausea, violent vomiting, bloody diarrhea and tenesmus, thirst, dilation of the pupils, conjunctivitis, shivering, and fever. Extra features of poisoning are mucosal damage, gastrointestinal bleeding, edema, renal failure, and vascular collapse. In severe poisoning, convulsions may precede death.
According to scientific procedure with nonhuman primates and a small number of human nonfatal poisoning cases (Associated Press, 2008; Poli et al., 2007), symptoms of inhalational exposure are respiratory distress, cough, fatigue, fever, and arthralgias that could come after by inflammation of airways, pneumonia, kidney failure, and coma. Based on animal data and human poisoning incidents, the lethal dose is estimated to be 1-20 mg/kg (3-10 castor beans, depending on size, moisture content as well as on the degree of mastication) by the oral route and 5-10 Î¼g/kg by injection or inhalation. For a general discussion on ricin toxicology and poisoning as relevant to humans, see the review by Audi et al. (2005).
The well-known allergy to castor bean and derived industrial products is due to proteinaceous allergens and not to ricin. Szalai et al. (2005), however, have delineated a series of allergic process among researchers working with ribosome RIPs, including ricin.
With a few exceptions, all reported human poisoning cases were due to swallowing of seeds of the castor plant, although death was exceptional (Audi et al., 2005; Rauber and Heard, 1985). Lufti (1935) narrated a fatal castor bean poisoning case. Two hours after ingesting 15-20 seeds, a man started to have nausea and abdominal pains, vomited, and later enduring diarrhea set in. The patient died as a result of complications of circulatory disorders, nephritis, and uremia 13 days after ingestion. Autopsy revealed that the kidneys, liver, heart, and spleen were hemorrhagic, necrotic, and inflamed.
Treatment consisting of ipecac-induced emesis, charcoal, and, to inhibit hemolysis, alkalinization of urine was given to children aged 7-12 ingesting one or two castor beans (Rauber and Heard, 1985). Except for mild diarrhea, no symptoms of castor bean toxicity were noticed. The same authors reported a suicide attempt of a patient ingesting at least 24 chopped castor beans. Following treatment consisting of induced emesis and gastroscopic removal of the bean particles, the patient was asymptomatic. Another recent case involved a young adult who ingested about a dozen castor beans, some of them chewed (Aplin and Eliseo, 1997). Four hours after ingestion, the patient started to feel ill, had severe abdominal pains, and vomited. The patient was given intravenous fluid, antiemetics, and charcoal 6 h after ingestion. After a mild hypokalemia (2.8 Î¼g/ml) on the second day, recovery was complete by the third day.
Kopferschmitt et al. (1983) portrayed a case of a 21-year-old man ingesting 30 castor beans (some masticated) to intentionally kill himself. Symptoms typical of ricin poisoning were observed. Radioimmunological analysis for ricin showed first-day plasma level of 1.5 ng/ml; the toxin appeared in the urine only on the third day (0.3 ng/ml). Treatment involved saline and glucose infusions and the patient recovered.
Another late case involved a young patient who tried to commit suicide by subcutaneously injecting an extract of castor beans (Targosz et al., 2002). The main symptoms presented 36 h after the injection were fatigue, nausea, dizziness, compression of chest, abdominal pain and muscular pain of extremities with numbness, anuria, tachycardia, hypotension, and metabolic acidosis. At the site of the needle insertion, suggillation and edema were seen. Bloody diarrhea and hemorrhagic diathesis as well as liver, kidney, cardiovascular and respiratory systems failure also developed. In spite of symptomatic intensive care, fatal asystolic arrest ensued 44 h after the injection of the poison.
The politically motivated murder of the Bulgarian playwright Georgi Markov is the most widely known fatal ricin poisoning case (Crompton and Gall, 1980). Ricin, which actually could not be directly identified, was dispensed in a small, hollow metallic sphere and probably shot from a modified umbrella into the right thigh of the victim. The amount of ricin in the sphere could be no more than 1 mg. Within a few hours, the victim was in pain, and the wound became inflamed. The following day, he had a high fever and vomited. Later, his blood pressure and temperature fell, the pulse rate rose to 160 beats/min, the white cell count was 26,300/ml, and renal tubular necrosis set in. Death was due to cardiac arrest on the third day after the injury. Autopsy revealed interstitial hemorrhages in the intestines, testicles, pancreas, and inguinal glands. Microscopy showed myocardial hemorrhages.
Distribution, metabolism, and excretion
I-Labeled ricin injected either intravenously or intraperitoneally was spread in various tissues, accumulating in the spleen, kidneys, heart, liver, and thymus (Fodstad et al., 1976). Urinary excretion of radioactive degradates, but not intact ricin, peaked 5-7 h after injection and was complete within 10-20 h. Because the intact toxin is resistant to proteolytic enzymes in vitro but the separated chains are considerably more vulnerable, it has been suggested that degradation in tissues occurs after reduction of the (disulfide bond) ASSB disulfide bridge and separation of the chains.
In a nasal-inhalation study with mice, ricin was initially agglomerated in the lungs with an approximately 40 h half-life but quickly distributed into the trachea (especially the associated thyroid) and the gastrointestinal tract; little evidence of systematic propagation of the toxin was observed at this inhaled dose (Doebler et al., 1995). The size of the aerosolized particles affects the distribution and toxicity of the toxin: inhalational exposure of mice to ricin particles of 1 Î¼m (at 4.5 * LD50 dose) outcome was 100% mortality by 72 h, while for toxin particles >5 Î¼m (at 3.7 * LD50 dose determined of the 1- Î¼m particle) no mortality was perceived (Roy et al., 2003; see also Griffiths et al., 2007).
This study also showed that the 1 Î¼m particles accumulated mainly in the lungs (60%), while larger particles deposited higher in the airways, that is in the trachea (80%). Comparing different mice strains, Godal et al. (1984) established that more sensitive strains concentrated higher amounts of ricin in their liver, spleen, and kidneys. The liver was rich in modified ricin and also in dissociated and modified A chains. Considerable amounts of the toxin accumulated in the adrenal cortex and bone marrow as well. In a subsequent phase I study with human cancer patients, the largest intravenous ricin dose with tolerable side effects (nausea and muscular pain) was 18 Î¼g/m2 with initial plasma concentration of about 25 ng/ml; concomitant anti-ricin antibody formation was also demonstrated.
Cook et al. (2006) have utilize an amplified ELISA (Enzyme-linked immunosorbent assay) with a limit of sensitivity of about 200 pg/ml to compare the tissue distribution of sublethal doses of crude ricin toxin (50% ricin content) following pulmonary and oral dosing to rats (250 g average weight). After pulmonary instillation of 0.8 Î¼g crude ricin, the total ricin content of the lung increased from 11.4 ng at 24 h to 40.9 ng at 48 h (corresponding to 7.12-11.38 ng/g tissue). However, the amount of ricin recovered from this tissue was 1-5% of the original dose. Alternatively, 24 h after an oral dose of 2 mg crude ricin per rat (8 mg/kg), the toxin was accumulated in the liver, spleen, gastrointestinal tract, and kidney (corresponding to 9.5, 31.7, 39.4, and 10.9 ng/g tissue, respectively). In this case, the total recovered amount was below 1% of the administered dose. It is also noteworthy, that blood, typically used for diagnostic purposes, contained only 1.4 ng/ml ricin.
Cause of death
The exact cause of death is unknown and probably varies with route of intoxication. Ingesting the toxin results in ulceration and hemorrhage of the stomach and small intestine mucosa, necrosis of the mesenteric lymphatics, liver necrosis, nephritis, and splenitis. Resultant loss of fluid and electrolytes may lead to hypotension, tachycardia, dehydration, cyanosis, and vascular collapse. Injection of the toxin may lead to severe local lymphoid necrosis, gastrointestinal hemorrhage, liver necrosis, diffuse nephritis, and diffuse splenitis. High doses administered intravenously in laboratory animals are associated with disseminated intravascular coagulation, and it has been suggested that hepatocellular and renal lesions result from vascular disturbances induced by the toxin rather than a direct effect of the toxin itself. Early studies clearly established that intravenous administration of ricin to rats results in diffuse damage to Kupffer cells within 4 hours, followed by endothelial cell damage, formation of thrombi in the liver vasculature, and finally, hepatocellular necrosis. In mice, rats, and primates, high doses by inhalation apparently produce lethal pulmonary damage, probably due to hypoxemia resulting from massive pulmonary edema and alveolar flooding.
Table 2. Clinical symptoms and progression of disease associated with ricin poisoning, by route of exposure.
Route of Exposure
Common Clinical Symptoms
Rapid onset of irritation of nose and throat;
Respiratory distress possibly leading to respiratory failure; Dyspnea (difficulty breathing); Pulmonary edema; Flu-like symptoms of fever, weakness, nausea, myalgia (muscle pain), or arthralgias (aches and pains in joints).
Nausea, vomiting, diarrhea, abdominal cramping and pain ;
Moderate to severe:
Persistent vomiting and voluminous diarrhea-bloody or non-bloody;
Dehydration and hypovolemic shock;
Hepatic and renal failure possible;
Mild hemolysis (not requiring blood transfusions); Liver and kidney dysfunction.
Flu-like symptoms with fatigue and myalgias; Local necrosis of muscles and regional lymph nodes at injection site;
Pain at injection site; Weakness, fever, and/or vomiting; Shock; Multi-organ failure.
Sources: Centers for Disease Control and Prevention, "Interim Ricin Response Plan (Draft). February 6, 2004; NIOSH Emergency Response Card: Ricin. February 18, 2004. Available at: http://www.bt.cdc.gov/ agent/ricin/erc9009-86-3.asp.