Ricin

Ricin (pronEng|ˈraɪ sɨn) is a protein toxin that is extracted from the castor bean ("Ricinus communis").

The U.S. Centers for Disease Control (CDC) gives a possible minimum figure of 500 micrograms (about the size of a grain of salt) Fact|date=May 2008 for the lethal dose of ricin in humans if exposure is from injection or inhalation. [http://www.bt.cdc.gov/agent/ricin/facts.asp http://www.bt.cdc.gov/agent/ricin/facts.asp] ]

Toxicity

Ricin is poisonous if inhaled, injected, or ingested, acting as a toxin by the inhibition of protein synthesis. While there is no known antidote, the US military has developed a vaccine. [ [http://www.dcmilitary.com/dcmilitary_archives/stories/090105/36813-1.shtml http://www.dcmilitary.com/dcmilitary_archives/stories/090105/36813-1.shtml] ] Symptomatic and supportive treatment is available. Long term organ damage is likely in survivors. Ricin causes severe diarrhea and victims can die of shock. Abrin is a similar toxin.It is estimated by the United States Department of Cultural and Biological Society in service to the United States Biological and Technological threat survey that to cover 25 km² area with 50% toxicity, about 1 metric ton of ricin is required.

Deaths caused by ingestion of castor oil plant seeds are rare. [cite journal |author=Aplin PJ, Eliseo T |title=Ingestion of castor oil plant seeds |journal=Med. J. Aust. |volume=167 |issue=5 |pages=260–1 |year=1997 |pmid=9315014 |doi=] Eight beans are considered toxic for an adult. [cite journal |author=Wedin GP, Neal JS, Everson GW, Krenzelok EP |title=Castor bean poisoning |journal=The American journal of emergency medicine |volume=4 |issue=3 |pages=259–61 |year=1986 |pmid=3964368|doi=10.1016/0735-6757(86)90080-X] A solution of saline and glucose has been used to treat ricin overdose. [cite journal |author=Kopferschmitt J, Flesch F, Lugnier A, Sauder P, Jaeger A, Mantz JM |title=Acute voluntary intoxication by ricin |journal=Human toxicology |volume=2 |issue=2 |pages=239–42 |year=1983 |pmid=6862467 |doi=] The case experience is not as negative as popular perception would indicate. [cite journal |author=Rauber A, Heard J |title=Castor bean toxicity re-examined: a new perspective |journal=Veterinary and human toxicology |volume=27 |issue=6 |pages=498–502 |year=1985 |pmid=4082461 |doi=]

Biochemistry

Ricin is classified as a type 2 ribosome inactivating protein (RIP). Whereas Type 1 RIPs consist of a single enzymatic protein chain, Type 2 RIPs, also known as holotoxins, are heterodimeric glycoproteins. Type 2 RIPs consist of an A chain that is functionally equivalent to a Type 1 RIP, covalently connected by a single disulfide bond to a B chain that is catalytically inactive, but serves to mediate entry of the A-B protein complex into the cytosol. Both Type 1 and Type 2 RIPs are functionally active against ribosomes in vitro, however only Type 2 RIPs display cytoxicity due to the lectin properties of the B chain. In order to display its ribosome inactivating function, the ricin disulfide bond must be reductively cleaved. [cite journal |author=Wright HT, Robertus JD |title=The intersubunit disulfide bridge of ricin is essential for cytotoxicity |journal=Arch Biochem Biophys |pages=280–4 |year=1987 |volume=256 |doi=10.1016/0003-9861(87)90447-4]

tructure

The tertiary structure of ricin was shown to be a globular, glycosylated heterodimer of approximately 60-65 kDA. [5] Ricin toxin A chain (RTA) and ricin toxin B chain (RTB) are of similar molecular weight, approximately 32 kDA and 34 kDA respectively.
* Ricin A Chain is an N-glycoside hydrolase composed of 267 amino acids. [cite journal |author=Olnes S, Pihl A |title=Different biological properties of the two constituent peptide chains of ricin, a toxic protein inhibiting protein synthesis |journal=Biochemistry |volume=12 | pages=3121–26 |year=1973 |doi=10.1021/bi00740a028] It has three structural domains with approximately 50% of the polypeptide arranged into alpha-helices and beta-sheets. [cite journal |author=Weston SA, Tucker AD, Thatcher DR, et al. |title=X-ray structure of recombinant ricin A-chain at 1.8 A resolution |journal=J Mol Biol | volume=244 |pages=410–22 |year=1994 |doi=10.1006/jmbi.1994.1739] The three domains form a pronounced cleft that is the active site of RTA.
* Ricin B Chain is a lectin composed of 262 amino acids that is able to bind terminal galactose residues on cell surfaces. [cite journal |author=Wales R, Richardson PT, Robers LM, Woodland HR, et al. |title=Mutational analysis of the galactose binding ability of recombinant ricin b chain |journal=J Biol Chem |volume=266 | pages=19172–79 |year=1991] RTB form a bilobal, barbell-like structure lacking alpha-helices or beta-sheets where individual lobes contain three subdomains. At least one of these three subdomains in each homologous lobe possesses a sugar-binding pocket that gives RTB its functional character.

Many plants such as barley have the A chain but not the B chain. People do not get sick from eating large amounts of such products, as ricin A is of extremely low toxicity as long as the B chain is not present.

Entry into the Cytosol

The ability of ricin to enter the cytosol depends on hydrogen bonding interactions between RTB amino acid residues and complex carbohydrates on the surface of eukaryotic cells containing either terminal N-acetyl galactosamine or beta-1,4-linked galactose residues. Additionally, the mannose-type glycans of ricin are able to bind cells that express mannose receptors. [cite journal | author=Magnusson AS, Kjeken R, Berg T | title=Characterization of two distinct pathways of endocytosis of ricin by rat liver endothelial cells | journal=Exp Cell Res | year=1993 | volume=205 | pages=118–25 | doi=10.1006/excr.1993.1065] Experimentally, RTB has been shown to bind to the cell surface on the order of 10⁶-10⁸ ricin molecules per cell surface. [cite journal | author=Sphyris N, Lord JM, Wales R, et al. | title=Mutational analysis of the ricinus lectin b-chains: Galactose-binding ability of the gamma subdomain of ricinus communis agglutin b-chain | journal=J Biol Chem | year=1995 | volume=270 | pages=20292–97 | doi=10.1074/jbc.270.35.20292 | pmid=7657599]

The profuse binding of ricin to surface membranes allows internalization with all types of membrane invaginations. Experimental evidence points to ricin uptake in both clathrin-coated pits, as well as clathrin-independent pathways including caveolae and macropinocytosis. [cite journal | author=Moya M, Dautry-Varsat A, Goud B, et al. | title=Inhibition of coated pit formatin in Hep2 cells blocks the cytotoxicity of diptheria toxin but not that of ricin toxin | journal=J Cell Biol | year=1985 | volume=101 | pages=548–59 | doi=10.1083/jcb.101.2.548 | pmid=2862151] [cite journal | author=Nichols, BJ, Lippincott-Schwartz J | title=Endocytosis without clathrin coats | journal=Trends Cell Biol | year=2001 | volume=11 | pages=406–12 | doi=10.1016/S0962-8924(01)02107-9] Vesicles shuttle ricin to endosomes that are delivered to the Golgi apparatus. The active acidification of endosomes are thought to have little effect on the functional properties of ricin. Because ricin is stable over a wide pH range, degradation in endosomes or lysosomes offer little or no protection against ricin.cite journal | author=Lord MJ, Jolliffe NA, Marsden CJ, et al. | title=Ricin Mechanisms of Cytotoxicity | journal=Toxicol Rev | year=2003 | volume=22 | issue=1 | pages=53–64 | doi=10.2165/00139709-200322010-00006] Ricin molecules are thought to follow retrograde transport through the Golgi and enter the endoplasmic reticulum (ER).

For ricin to function cytotoxically, RTA must be reductively cleaved from RTB in order to release a steric block of the RTA active site. Currently, it is unknown whether this takes place in the ER or in the cytosol. It is speculated that within the ER, RTA utilizes the endoplasmic reticulum-associated protein degradation (ERAD) pathway that exists to eject misfolded proteins to the cytosol. [cite journal | author=Roberts LM, Smith DC | title=Ricin: the endoplasmic reticulum connection | journal=Toxicon | year=2004 | volume=44 | pages=469–72 | doi=10.1016/j.toxicon.2004.07.002] Chaperones participating in ERAD may recognize RTA as misfolded native protein and translocate it into the cytosol. Additionally, RTA resists degradation by ubiquitination that often occurs with misfolded proteins by maintaining a low content of lysine residues, the usual attachment sites for ubiquitin. [cite journal | author=Deeks ED, Cook JP, Day PJ, et al. | title=The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol | journal=Biochemistry | year=2002 | volume=41 | pages=3405–13 | doi=10.1021/bi011580v] In the cytosol, RTA is free to exert its toxicity on ribosomes.

Ribosome Inactivation

Study of the N-glycosidase activity of ricin was pioneered by Endo and Tsurugi [cite journal | author=Endo Y, Tsurugi K | title=RNA N-glycosidase activity of ricin A-chain: mechanism of action of the toxic lectin ricin on eukaryotic ribosomes | journal=J Biol Chem | year=1987 | volume=262 | pages=8128–30] who showed that RTA cleaves a glycosidic bond within the large rRNA of the 60S subunit of eukaryotic ribosomes. They subsequently showed RTA specifically and irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 (A4324) within the 28S rRNA, but leaves the phosphodiester backbone of the RNA intact. [cite journal | author=Endo Y, Tsurugi K | title=The RNA N-glycosidase activity of ricin A chain | journal=J Biol Chem | year=1998 | volume=263 | pages=8735–9] The ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes. The sequence, 5’-AGUACGAGAGGA-3’, termed the sarcin-ricin loop, is important in binding elongation factors during protein synthesis. [ cite journal | author=Sperti S, Montanaro L, Mattioli A, et al. | journal=Biochem J | year=1973 | volume=136 | pages=813–5
unused_data = |Inhibition by ricin of protein synthesis in vitro: 60S ribosomal subunit as the target of the toxin] The depurination event rapidly and completely inactivates the ribosome, resulting in toxicity from inhibited protein synthesis. A single RTA molecule in the cytosol is capable of depurinating approximately 1500 ribosomes per minute.

Depurination reaction

Within the active site of RTA, there exist several invariant amino acid residues involved in the depurination of ribosomal RNA. Although the exact mechanism of the event is unknown, key amino acid residues identified include tyrosine at positions 80 and 123, glutamic acid at position 177, and arginine at position 180. In particular, Arg180 and Glu177 have been shown to be involved in the catalytic mechanism, and not substrate binding, with enzyme kinetic studies involving RTA mutants. The model proposed by Mozingo and Robertus, [ cite journal | author=Monzingo AF, Robertus JD | title=X-ray analysis of substrate analogs in the ricin A-chain active site | journal=J Mol Biol | year=1992 | volume=244 | pages=410–22 | doi=10.1006/jmbi.1994.1739] , based x-ray structures, is as follows:

1. Sarcin-ricin loop substrate binds RTA active site with target adenine stacking against tyr80 and tyr123.
2. Arg180 is positioned such that it can protonate N-3 of adenine and break the bond between N-9 of the adenine ring and C-1’ of the ribose.
3. Bond cleavage results in an oxycarbonium ion on the ribose, stabilized by Glu177.
4. N-3 protonation of adenine by Arg180 allows deprotonation of a nearby water molecule.
5. Resulting hydroxyl attacks ribose carbonium ion.
6. Depurination of adenine results in a neutral ribose on an intact phosphodiester RNA backbone.

Manufacture

Ricin is easily purified from castor-oil manufacturing waste. The aqueous phase left over from the oil extraction process is called as waste mash. It contains about 5-10% of ricin by weight. Separation of this only requires simple chromatographic techniques.

Patented extraction process

The process for extracting ricin is well-known, and for example described in a patent. ["Preparation of Toxic Ricin", US patent|3060165, assigned to the U.S. Secretary of the Army, inventors: Harry L. Craig, O.H. Alderks, Alsoph H. Corwin, Sally H. Dieke, and Charlotte Karel (granted October 23, 1962)] The described extraction method is very similar to the preparation of soy protein isolates.

The patent was removed from the United States Patent and Trademark Office (USPTO) database sometime in 2004, but is still available online through international patent databases. [http://v3.espacenet.com/origdoc?DB=EPODOC&IDX=US3060165&QPN=US3060165 Harry L. Craig, O.H. Alderks, Alsoph H. Corwin, Sally H. Dieke, and Charlotte Karel, US Patent 3,060,165, "Preparation of Toxic Ricin", granted October 23, 1962] ] [ [http://cryptome.org/ricin-patent.htm Ricin Patent ] ] Modern theories of protein chemistry cast doubt on the effectiveness of the methods disclosed in the patent. [http://www.globalsecurity.org/org/nsn/nsn-040723.htm http://www.globalsecurity.org/org/nsn/nsn-040723.htm] ]

Potential medicinal use

Some researchers have speculated about using ricins in the treatment of cancer, as a so-called "magic bullet" to destroy targeted cells: [cite journal |author=Lord MJ, Jolliffe NA, Marsden CJ, "et al" |title=Ricin. Mechanisms of cytotoxicity |journal=Toxicological reviews |volume=22 |issue=1 |pages=53–64 |year=2003 |pmid=14579547 |doi=] Ricin could be linked to a monoclonal antibody to target malignant cells recognized by the antibody. The major problem with ricin is that its native internalization sequences are distributed throughout the protein. If any of these native internalization sequences are present in a therapeutic, then the drug will be internalized by, and kill, untargeted epithelial cells as well as targeted cancer cells.

Some researchers hope that modifying ricin will sufficiently lessen the likelihood that the ricin component of these immunotoxins will cause the wrong cells to internalize it, while still retaining its cell-killing activity when it is internalized by the targeted cells. Generally, however, ricin has been superseded for medical purposes by more practical fragments of bacterial toxins, such as diphtheria toxin, which is used in denileukin diftitox, an FDA-approved treatment for leukemia and lymphoma. No approved therapeutics contain ricin.

A promising approach is also to use the non-toxic B subunit as a vehicle for delivering antigens into cells thus greatly increasing their immunogenicity. Use of ricin as an adjuvant has potential implications for developing mucosal vaccines.

Use as a chemical/biological warfare agent

The United States investigated ricin for its military potential during the First World War. At that time it was being considered for use either as a toxic dust or as a coating for bullets and shrapnel. The dust cloud concept could not be adequately developed, and the coated bullet/shrapnel concept would violate the Hague Convention of 1899. The war ended before it was weaponized.

During the Second World War the United States and Canada undertook studying ricin in cluster bombs. Though there were plans for mass production and several field trials with different bomblet concepts, the end conclusion was that it was no more economical than using phosgene. This conclusion was based on comparison of the final weapons rather than ricin's toxicity (LCt₅₀ ~40 mg·min/m³). Ricin was given the military symbol W or later WA. Interest in it continued for a short period after the Second World War, but soon subsided when the U.S. Army Chemical Corps began a program to weaponize sarin.

The Soviet Union also had ricin. There were speculations that KGB even used it outside of the Soviet bloc; however, this was never proven. In 1978, the Bulgarian dissident Georgi Markov was assassinated by Bulgarian secret police who surreptitiously 'shot' him on a London street with a modified umbrella using compressed gas to fire a tiny pellet contaminated with ricin into his leg. [cite web |url=http://www.cnn.com/2003/WORLD/europe/01/07/terror.poison.bulgarian/ |title=Ricin and the umbrella murder |accessdate=2008-03-15 |format= |work= ] He died in a hospital a few days later; his body was passed to a special poison branch of the British Ministry of Defence (MOD) that discovered the pellet during an autopsy. The prime suspects were the Bulgarian secret police: Georgi Markov had defected from Bulgaria some years previously and had subsequently written books and made radio broadcasts which were highly critical of the Bulgarian communist regime. However, it was believed at the time that Bulgaria would not have been able to produce the pellet, and it was also believed that the KGB had supplied it. The KGB denied any involvement although high-profile KGB defectors Oleg Kalugin and Oleg Gordievsky have since confirmed the KGB's involvement. Earlier, Soviet dissident Aleksandr Solzhenitsyn also suffered (but survived) ricin-like symptoms after a 1971 encounter with KGB agents. [D.M. Thomas, "Alexander Solzhenitsyn: A Century in His Life," 368-378]

Despite ricin's extreme toxicity and utility as an agent of chemical/biological warfare, it is extremely difficult to limit the production of the toxin. Under both the 1972 Biological Weapons Convention and the 1997 Chemical Weapons Convention, ricin is listed as a schedule 1 controlled substance. Despite this, more than 1 million metric tonnes of castor beans are processed each year, and approximately 5% of the total is rendered into a waste containing high concentrations of ricin toxin. [http://www.ansci.cornell.edu/plants/toxicagents/ricin/ricin.html http://www.ansci.cornell.edu/plants/toxicagents/ricin/ricin.html] ]

To put ricin used as a weapon into perspective, it is worth noting that as a biological weapon or chemical weapon, ricin may not be considered very powerful in comparison with other agents such as botulinum or anthrax. Hence, a military willing to use biological weapons "and" having advanced resources would rather use either of the latter instead. Ricin is easy to produce, but is not as practical nor likely to cause as many casualties as other agents. Ricin is inactivated (the protein changes structure and becomes less dangerous) much more readily than anthrax spores, which may remain lethal for decades. (Jan van Aken, an expert on biological weapons explained in an interview with the German magazine Der Spiegel that he judges it rather reassuring that Al Qaeda experimented with ricin as it suggests their inability to produce botulin or anthrax.)

The major reason it is dangerous is that there is no specific antidote, and that it is very easy to obtain (the castor bean plant is a common ornamental, and can be grown at home without any special care). There have been several reported incidents where ricin has been involved with infanticide where small children have been tricked into eating castor beans because of their striking resemblance to chocolate covered coffee beans. Ricin is actually several orders of magnitude less toxic than botulinum or tetanus toxin, but those are more difficult to obtain.

Detected ricin incidents

1978 assassination of Georgi Markov

On 7 September 1978 the Bulgarian dissident Georgi Markov was stabbed in the leg in public on Waterloo Bridge in the middle of London by a man using a weapon built into an umbrella. The weapon embedded a small pellet in Markov's leg which contained ricin. Markov died four days later.Fact|date=March 2008

2000 discovery in Irvine, California home

On 2 March, 2000, following the suicide of Dr. Larry C. Ford three days after the attempted murder of his business partner, police searching his Irvine, California home discovered ricin, a blowgun, and darts in a plastic bag in the family room. Assault rifles and C-4 plastic explosives were found in containers buried next to his swimming pool. In refrigerators at his home and office, next to the salad dressing and employee lunches, were 266 bottles and vials of pathogens. Among them were the bacterial agents of cholera ("vibrio cholerae"), botulism ("clostridium botulinum"), and typhoid ("salmonella typhi"). City officials closed an elementary school and evacuated several homes in the neighborhood while they performed a thorough search of Ford's home.

The searchers did not find anthrax and the fear of what remained to be found, along with many other questions, set off investigations spanning from Beverly Hills to South Africa.

2003 arrests in Britain

On 5 January, 2003 the Metropolitan Police raided a flat in north London and arrested six Algerian men whom they claimed were manufacturing ricin as part of a plot for a poison attack on the London Underground. No ricin was recovered as a result of this raid.

2003 envelope in South Carolina

In 2003, a package and letter sealed in a "ricin-contaminated" envelope was intercepted in Greenville, South Carolina, at a United States Postal Service processing center. [ [http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5246a5.htm Investigation of a Ricin-Containing Envelope at a Postal Facility - South Carolina, 2003 ] ]

2003 White House mail

Ricin was detected in the mail at the White House in Washington, D.C. in November 2003. The letter containing it was intercepted at a mail handling facility off the grounds of the White House, and it never reached its intended destination. The letter contained a fine powdery substance that later tested positive for ricin. Investigators said it was low potency and was not considered a health risk. This information was not made public until February 3, 2004, when preliminary tests showed the presence of ricin in an office mailroom of U.S. Senate Majority Leader Bill Frist's office. There were no signs that anyone who was near the contaminated area developed any medical problems. Several Senate office buildings were closed as a precaution.

2006 home in Richmond, Virginia

In January 2006, ricin was found in a home in suburban Richmond, Virginia in the form of mashed castor beans. Although the suspect, Chetanand Sewraz, was allegedly isolating the toxin to kill his estranged wife, and not for some form of bioterrorism, it nonetheless highlighted the ease with which ricin toxin can be made. [http://wtvr.com/Global/story.asp?S=4457615 http://wtvr.com/Global/story.asp?S=4457615] ] [http://wtvr.com/Global/story.asp?S=4457626 http://wtvr.com/Global/story.asp?S=4457626] ]

2008 hotel room in Las Vegas, Nevada

In February 2008, a man who stayed in a Las Vegas, Nevada motel room where ricin was found was taken to hospital in critical condition. The man, Roger Von Bergendorff, was hospitalized on February 14; however, the ricin was not found until February 27 when a relative retrieved his luggage because the motel had not been paid for two weeks. Firearms and an "anarchist type textbook" were found in the same motel room where several vials of ricin were found, police reported. According to Las Vegas 8 Television news, police noted the ricin section of the textbook was highlighted. [ [http://www.cnn.com/2008/US/02/29/ricin.hotel/index.html Police: Man in critical condition after exposure to ricin - CNN.com ] ] [http://news.yahoo.com/s/ap/20080229/ap_on_re_us/motel_hazardous_material;_ylt=AtfdNqvbxuM_cNBPbBipVYCs0NUE] On March 3rd, FBI agents searched a Riverton, Utah house and several storage lockers in West Jordan, Utah linked to Bergendorff, but did not find any traces of ricin. [ [http://edition.cnn.com/2008/US/03/02/motel.ricin.ap/index.html FBI searches home, storage units after ricin find - CNN.com ] ] [ [http://origin.sltrib.com/ci_8428616 Riverton residents return to homes, but search for deadly toxin continues - Salt Lake Tribune ] ] [http://www.fox5vegas.com/news/15597214/detail.html Man Wakes From Ricin Coma - Local News Story - KVVU Las Vegas ] ] Bergdendorff awoke from a comatose condition on March 14th. He was questioned by police as to why he had such a large quantity of ricin. Subsequently, he was arrested on April 16 and charged with possession of a biological toxin and two weapons offenses. [ [http://news.yahoo.com/s/ap/20080416/ap_on_re_us/ricin_mystery Man at center of Las Vegas ricin case arrested, charged] ]

ee also

* 2001 anthrax attacks

References

External links

* [http://www.hort.purdue.edu/newcrop/afcm/castor.html Castor bean information] at Purdue University
* [http://www.phls.co.uk/topics_az/deliberate_release/pdf/ricin_guidelines.pdf ricin information] at Department of Health (United Kingdom)
* [http://www.ansci.cornell.edu/plants/toxicagents/ricin/ricin.html ricin information] at Cornell University
* [http://news.bbc.co.uk/2/hi/health/2837763.stm Medical research on ricin] at BBC
* [http://www.wood.army.mil/chmdsd/Army_Chemical_Review/pdfs/2004%20Apr/Kirby-Ricin-04-1.pdf Chemical Review] at United States Army
* [http://www.bt.cdc.gov/agent/ricin/ Ricin - Emergency Preparations] at CDC