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Home , Aflatoxin, Mycotoxin, Poison dart frog, Shiga toxin, Trichothecene

journal of applied biomedicine 12 (2014) 63–77

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journal homepage: http://www.elsevier.com/locate/jab

Review Article Military potential of biological

Xiujuan Zhang a, Kamil Kuča b,c,**, Vlastimil Dohnal c, Lucie Dohnalová d, Qinghua Wu e, Chu Wu a,* a College of Horticulture and Garden, Yangtze University, Jingzhou, Hubei 434023, People's Republic of b Biomedical Research Center, University Hospital Hradec Kralove, Czech Republic c Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Rokitanskeho 62, 500 03, Czech Republic d Department of Hygiene, Faculty of Military Health, University of Defence, Hradec Kralove, Czech Republic e National Reference Laboratory of Veterinary Drug Residues (HZAU), Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China

article info abstract

Article history: Toxins are produced by , and for defense or for predation. Most of the Received 27 November 2013 toxins specifically affect the mammalian by interfering with the transmission Received in revised form of nerve impulses, and such toxins have the potential for misuse by the military or terrorist 17 February 2014 organizations. This review discusses the origin, structure, and symptoms, transmis- Accepted 19 February 2014 sion, mechanism(s) of action, symptomatic treatment of the most important toxins and Available online 1 March 2014 derived from fungi, plants, marine animals, and , along with their potential for use in bioweapons and/or biocrime. Fungal and aflatoxins are Keywords: potent inhibitors of protein synthesis in most eukaryotes and have been used as biological Military toxins warfare agents. and are -derived toxins that prevent the elongation of Trichothecenes polypeptide chains. , anatoxin, and are marine-derived toxins that toxins bind to sodium channels in nerve and muscle tissue and cause muscle paralysis. Most bacterial Bioweapons toxins, such as botulinum and Shiga affect either the nervous system () or damage Toxicity membranes. , which are secreted by -dart are extremely potent cardiotoxic and neurotoxic steroidal . The aim of this review is to provide basic information to enable further understanding of these toxins and their potential military uses. Abbreviations: # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske AFB, aflatoxins B Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. STEC, Shiga -producing STX, saxitoxin TTX, tetrodotoxin BTX, batrachotoxins

* Corresponding author at: College of Horticulture and Garden, Yangtze University, Jingzhou, Hubei 434023, P.R. China. Tel.: +86 0716 8066260; fax: +86 0716 8066262. ** Corresponding author at: Department of Toxicology, Faculty of Military Health, University of Defence, Hradec Kralove, Czech Republic. Tel.: +420 603289166. E-mail addresses: [email protected], [email protected], [email protected] (K. Kuča), [email protected] (C. Wu). 1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.jab.2014.02.005 64 journal of applied biomedicine 12 (2014) 63–77

Trichothecene (T-2) Introduction Origin Many potent toxins are nature compounds produced by T-2 toxin as one of the primary members of type-A microorganisms, plants, and animals. T-2 toxin (also known trichothecenes is produced mainly by genus. The ‘‘’’) is one of the most toxic trichothecenes and is most important producer is Fusarium sporotrichioides, a sapro- thought to have been used in 1980s as a chemical weapon in phyte (Moss, 2002). T-2 toxin has been recognized as an Southeast (Desjardins, 2009). Aflatoxins are known to unavoidable contaminant in cereals such as , , have been weaponized by Iraq (so-called ‘‘cancer bombs’’) , , and feed (Guan et al., 2009). (Paterson, 2006). Most of bacterial toxins are large proteins that T-2 toxin is associated with several diseases in animals and affect either the nervous system (neurotoxins) or damage cell humans. Alimentary toxic aleukia (ATA), a typical disease for membranes. Ricin, another potent toxin named ‘‘compound human, is found to be associated primarily with the ingestion W’’ by the British military, as incorporated into a bomb; of moldy cereal infected with T-2 toxin (Joffe, 1974). Due to its however, the ‘‘W-bomb’’ has never actually used (Crompton high toxicity and stability, T-2 toxin is classified as biological and Gall, 1980). Marine-derived toxins (such as anatoxins, weapon (Kuca and Pohanka, 2010). saxitoxin (STX) and tetrodotoxin (TTX)) have the potential for use in terrorist-designed biological weapons, but are unlikely Chemical structure to pose a major threat in an open battlefield situation (Franz, Trichothecenes are tetracyclic sesquiterpenoid compounds 1996). The use of toxins as weapons may increase in the future harboring a C-12-C-13-epoxy group. The skele- because they are highly effective and can be used surrepti- ton (scirpene) comprises a pyran group, a C-12-C-13- tiously. ring, and a double (olefinic) bond at C-9-C-10. Trichothecenes Understanding the mechanism(s) underlying toxicity is the are grouped into four categories (A–D) based on their structural first step to developing medical countermeasures. Toxins fall characteristics. T-2 and HT-2 toxins belong to group A, which is into two broad categories, as follows: (a) neurotoxins, which characterized by a functional group other than a carbonyl at C- affect nervous system function (most often the peripheral 8 (Wu et al., 2010, 2011). The chemical structures of the nervous system) and the effect of which are typically trichothecenes are shown in Fig. 1. temporary and/or reversible; and (b) membrane-damaging Trichothecenes are nonvolatile compounds with a molec- toxins, which destroy or damage tissues or organs either ular weight between 250 and 550 Da. They are relatively directly or indirectly via the release of secondary mediators. insoluble in but highly soluble in organic solvents such The effects of membrane-damaging toxins are often not as acetone, ethyl acetate, chloroform, and dimethylsulfoxide. reversible (Franz, 1996). Trichothecene compounds are stable when exposed to air Understanding the global profile of these toxins is very and/or light, but they can be degraded by the presence of important for health and risk assessment; however, military bacteria and fungi. Trichothecenes are not inactivated by toxins have never been fully evaluated. The difference autoclaving, but are deactivated by strongly acidic or alkaline between conventional chemical weapons and biological conditions and by heating to 900 8F (482 8C) for 10 min or to warfare agents has been discussed by Madsen (2001). Bigalke 500 8F(2608C) for 30 min (Wannenmacher and Wiener, 1997; and Rummel (2005) and Anderson (2012) discussed the use of Sudakin, 2003). tirchothecenes, ricin, and botulinum neurotoxins and Dixit et al. (2005) reviewed many neurotoxins; however, little was Toxicity and symptoms mentioned regarding detailed mechanisms of action, trans- Trichothecenes are toxic to humans, animals, and plants mission and possible treatments. Pita (2009) authored an (Wannenmacher and Wiener, 1997). Their varies interesting article about the use of toxins as meapons from with animal and/or the route of administrations. The Æ World War I to the present day. LD50 value of T-2 toxin in rats and rabbits is 0.85 0.03 and Here, we will discuss the major toxins derived from fungi, 1.10 Æ 0.08 mg/kg body weight, respectively (Chan and Gentry, plants, marine animals and frogs along with their venoms in 1984). Acute exposure to weapons containing trichothecene spite of their origin, structure, toxicity and symptoms, is a major concern for those working in the filed of transmission, mechanism(s) of action, symptomatic treat- military medicine (Wannenmacher and Wiener, 1997). Early ment, and their potential use for bioweapons. symptoms and signs included severe nausea, vomiting, superficial skin discomfort (burning sensation), lethargy, weakness, dizziness, and loss of coordination. Diarrhea (first Fungal toxins watery brown and later grossly bloody) begins within minutes to hours after exposure. From 3 to 12 h post-exposure, the Fungal toxins are low molecular weight compounds victim suffers dyspnea, coughing, sore mouth, bleeding gums, (<1000 g/mol) produced by fungi. The potential use of fungal epistaxis, hematemesis, abdominal pain, and central chest toxins as weapons or ‘‘expressions of discontent’’ is taken very pain. The exposed cutaneous areas become red, tender, seriously because they can be used by governments or by small swollen, painful, or pruritic. Small or large vesicles and bullae groups of individuals. For example, an individual could use such might form, and petechiae, ecchymoses, and black leathery a toxin to carry out a revenge attack on an employer (Paterson, areas of may appear. After death, the necrotic areas 2006). Indeed, trichothecene mycotoxins and aflatoxins are slough easily when the corpse is moved (Wannenmacher and thought to have been used as agents. Wiener, 1997). journal of applied biomedicine 12 (2014) 63–77 65 [(Fig._1)TD$IG]

Fig. 1 – Chemical structure of trichothecenes (examples of groups A–D).

Transmission Partial inhibition of RNA (McLaughlin et al., 1977) and DNA Trichothecene mycotoxins can be inhaled, ingested, or synthesis (Thompson and Wannemacher, 1990) by trichothe- absorbed through the skin (Wannenmacher and Wiener, cene mycotoxins was observed in various cell cultures. 1997) but cannot be transmitted from person-to-person, although trichothecenes can be dispersed in the air or mixed Potential use as a bioweapon or in biocrime with or beverages. The toxin can also be delivered by Because of their antipersonnel properties, ease of large-scale mortars, artillery, free rockets, and aerial bombs. There is a production, and proven delivery via various aerial dispersal distinct possibility that T-2 toxin and its metabolites can be systems, trichothecenes (especially T-2 toxin) show excellent transferred to edible tissues in animals that have ingested potential for weaponization (Wannenmacher and Wiener, contaminated feed (Chi et al., 1978; Yoshizawa et al., 1981). 1997). Indeed, it is suspected that mycotoxins were used as chemical weapons in northern Laos, Southeast Asia, more Mechanism of action than 30 years ago (Rosen and Rosen, 1982; Mirocha et al., 1983; The primary toxic effects of trichothecenes are due to their Desjardins, 2009). People living in bombed areas showed potent inhibition of eukaryotic protein synthesis (McLaughlin symptoms of trichothecene mycotoxin poisoning, and et al., 1977; Thompson and Wannemacher, 1984). Trichothe- reported rain that left sticky yellow drops that dried up very cenes are amphophilic molecules and may, therefore, also act quickly. In other cases, it was reported as a cloud of yellow on the cell membranes. Once trichothecenes cross the plasma dust, which subsequently contaminated the environment in membrane, they interact with a number of targets, including the form of rain. The toxic agent was an oily yellow liquid with and mitochondria (McLaughlin et al., 1977; Pace a relatively large droplet size that sounded like rain when it et al., 1988). The toxins interfere with the active site within struck the ground, vegetation, and the roofs of houses; thus it on ribosomes and inhibit the initiation (I), was known as ‘‘Yellow Rain’’ (Tucker, 2001). elongation (E) or termination (T) steps of protein synthesis However, researchers did not agree on the composition of (Wannenmacher and Wiener, 1997; Rocha et al., 2005). Thus ‘‘Yellow Rain’’. Australian researchers examined 'Yellow Rain' they are known as I-type, E-type or T-type toxins. samples from Southeast Asia and found that the samples did I-type trichothecenes inhibit either the formation of the 80S not contain any toxic compounds; rather, they contained complex or the function of the intact . E-type or T- yellow pollen grains together with a small amount of binder type trichothecenes act by binding to the ribosomal peptidyl (Budiansky, 1983a,b). Therefore, by the end of 1983 to mid- transferase site on the 60S subunit and preventing peptide 1984, the commonly accepted opinion was that 'Yellow Rain' bond formation. Trichothecenes can trigger a ribotoxic stress was simply the excreta from honey bees (Budiansky, 1983c; response that actives c-Jun N-terminal kinase/p38 mitogen- Nowickej and Mfselson, 1984). activated protein kinases, components of a signaling cascade that induces and regulates cell survival in response Symptomatic treatment to stress (Kiessling, 1986; Shifrin and Andreson, 1999). Toxicity may be prevented by vigorous washing with soap and Trichothecene-related inhibition of RNA synthesis is probably water within 1 h of exposure which will clear the residues from secondary to the effect of the inhibition of protein synthesis. the skin. Contaminated clothing must be removed and 66 journal of applied biomedicine 12 (2014) 63–77

disposed of. Delayed mild skin symptoms can be treated with such as rainbow trout, cats, and ducklings. Tests involving calamine lotion, camphor, or other similar lotions, and various animal species and mammalian cells show that the activated can be used in cases of ingestion. Mild toxicity of AFG1 and AFB2 and AFG2 is approximately 50%, 20% respiratory symptoms can be treated with codeine, cough and 10%, respectively, of that of AFB1 (Agag, 2004). suppressants, or steam inhalation (Wu et al., 2010). Eyes Aflatoxins may also induce the development of human should be irrigated with copious amounts of saline. Solutions (HCC) (Busby and Wogan, 1984). containing 1% and 0.1 M NaOH are used Clinically, the main features of this syndrome are vomiting, for environmental decontamination (Yuen, 2001). convulsions, and coma. Hypoglycemia and elevated serum transaminase levels are the most consistent biochemical Aflatoxins abnormalities. Fatty degeneration in the and kidneys, and cerebral edema are noted in the majority of autopsy findings Origin (Agag, 2004). Aflatoxins are a group of highly toxic secondary metabolites The most extensive and widely publicized outbreak of produced mainly by species fungi (Wu et al., 2009). aflatoxin toxicosis was reported in Western India in 1974. The These toxins can be found mainly in cereals, oil seeds, tree illness was characterized by jaundice, rapidly developing nuts, , and milk. The most commonly occurring ones are ascites, portal hypertension, and a high mortality rate. aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), Extensive bile duct proliferation, periportal fibrosis, and and aflatoxin G2 (AFG1) in fungi cultures. Aflatoxins have been occasional multinucleated giant cells (typical of aflatoxin- demonstrated to produce primary in animals (Wu induced diseases in animals) were found upon histopathologic et al., 2009) and AFB1 are classified as group 1 by examination of liver specimens (Krishnamachari et al., 1975; the International Agency for Research on Cancer (IARC) (IARC, Robens and Richard, 1992). 2002). Transmission Chemical structure Aflatoxins can be directly ingested via contaminated of There are 18 different types of aflatoxins, the most common of plant origin, including maize, nuts and their associated which are AFB1, AFB2, AFG1, and AFG2 (Wu et al., 2009). Pure products. Aflatoxins can be transmitted from animal feed

AFB1 (C17H12O6) is a pale white-to-yellow crystalline, odorless into milk and milk products, including cheese and powdered solid. Aflatoxins are soluble in , chloroform, and milk, where they appear mainly as aflatoxin M1 (AFM1) (World acetonitrile. Structurally speaking, the double bond-containing Health Organization, 1979). Indeed, residues of aflatoxins may dihydrofuran moiety and constituents liked to the be present in the tissues of animals that consume contami- moiety are the mediators of their biological effects. Aflatoxins nated feed (World Health Organization, 1979). For example, a fluorescence strongly in light; AFB1 and AFB2 laying mash ration used to feed laying hens contained AFB1 at produce a blue fluorescence whereas AFG1 and AFG2 produce levels of 0.1, 0.2, and 0.4 parts per million (ppm) and the eggs green fluorescence (Reddy and Waliyar, 2000). The structures of from these hens contained measurable amounts of AFB1 AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 are shown in Fig. 2. (Jacobson and Wiseman, 1974).

Toxicity and symptoms Mechanism of action Not only are aflatoxins carcinogenic and teratogenic in Aflatoxins are carcinogenic and mutagenic because they form animals, they also cause impaired protein formation and a reactive epoxide at the 8, 9-position of the terminal furan ring induce coagulation, weight gain, and immunogenesis (Robens by (CYP) and its subsequent – and Richard, 1992). AFB1 is highly toxic (LD50,1 50 mg/kg body covalent binding to nucleic acids (Essigmann et al., 1982; weight) to most animal species, although it is extremely toxic Wu et al., 2009). The epoxide reacts with guanine bases within < (LD[(Fig._2)TD$IG] 50 1 mg/kg body weight) to highly susceptible species DNA and RNA, resulting depurination. The primary effect is to

Fig. 2 – Chemical structure of B1, B2, M1 and M2. journal of applied biomedicine 12 (2014) 63–77 67

inhibit DNA and protein synthesis in the most active tissues, Chemical structure including the liver, intestines, and . The damage Ricin is a globular, glycosylated heterodimer in which the ricin A to DNA can be mutagenic, typically involving a GC to AT and B chains are joined by a single disulphide bond (Fig. 3). The A mutation; ultimately, ensues, with HCC a chain is an N-glycoside hydrolase comprising 267 amino acids, common long-term consequence of exposure (Essigmann with approximately 50% of the polypeptide arranged into a- et al., 1982). helices and eight b-sheets. The B chain is a lectin composed of 262 amino acids. It forms a structure that lacks the a-helix and Potential use as a bioweapon and in biocrime eight b-sheets, and probably arose through duplication Aflatoxins are not the most toxic of weaponizable toxins and (Katzin et al., 1991; Musshoff and Madea, 2009). Ricin is stable their military value is questioned by a number of authorities under normal conditions, but can be inactivated by heating (Bennett and Klich, 2003; Paterson, 2006). However, they are above 80 8C (Roxas-Duncan and Smith, 2012). relatively easy to manufacture and can be used to commit enemy forces into protective gear, thereby reducing their Toxicity and symptoms combat effectiveness. To date, flatoxins have not been there The signs, symptoms, and severity of ricin poisoning vary used as bioweapons in terrorist attacks. according to the dose and route of exposure (Holterman, 2006). Typically, there is an asymptomatic period (hours or days

Symptomatic treatment depending on the dose) after oral consumption. The oral LD50 in Once exposed, there is a risk of future cancer and no rats and mice is 20–30 mg/kg body weight. Orally ingested ricin treatments are available to prevent this, thus avoidance of is 1000- to 10,000-fold less toxic than injected (intraperitoneal food containing aflatoxins is the primary work. Amphotericin (i.p.) and intravenous) or inhaled forms (Garber, 2008). Four-to- B and itraconazole may be helpful for the acute exposure (Wu eight castor oil seeds contain enough ricin to kill a child. et al., 2009). Transmission Ricin is not contagious and cannot spread from person-to- Plant toxins person via casual contact; however, it could be spread person- to-person if the powder were on the clothing of exposed Plant toxins comprise proteins that are very cheap and easy to individuals (Holterman, 2006). produce in large quantities in a low-technology environment. Mechanism of action Ricin The ricin B chain (a lectin) binds to galactose-containing glycoproteins and expressed on the surface of cells. Origin Once bound, ricin enters the cells by endocytosis and is Ricin is a heterodimeric protein present in the seeds of Abrus transported to the endosomes and then (via the Golgi) to the precatorius L. Although the castor oil plant originated in Asia and endoplasmic reticulum. From there, the ricin A chain is , it now grows in all temperate and subtropical regions translocated to the cytosol, where it removes an adenine base (Spivak and Hendrickson, 2005; Musshoff and Madea, 2009). It is from the 28S ribosomal RNA loop contained within the 60S relatively easy to purify ricin from the waste products of the ribosomal submit; this leads to the irreversible inactivation of castor oil manufacturing industry using organic solvents such protein synthesis and subsequent cell death (Audi et al., 2005; as hexanes and carbon tetrachloride; ricin is left behind in the Musshoff and Madea, 2009). mash. Every year more than one million tonsof castoff beans are produced, providing a convenient source of ricin (Doan, 2004; Potential use as a bioweapon and in biocrime Roxas-Duncan and Smith, 2012). Ricin is not considered an effective weapon of mass destruction; [(Fig._3)TD$IG] however, its potential as a weapon of terror cannot be ignored. During the Second World War, the British military called ricin ‘‘compound W’’ and developed a ‘‘W-bomb’’;however,itwas never used against soldiers or civilians. The well-publicized ‘‘umbrella murder’’ of the Bulgarian dissident Georgi Markov in London in 1978 is the first reported case of ricin used in political murder (Crompton and Gall, 1980). The US Centers for Disease Control and Prevention (CDC) has classified ricin as a Category B threat. Musshoff and Madea (2009) and Roxas-Duncan and Smith (2012) discuss more cases in which ricin has been used as a murder weapon or as an espionage tool.

Symptomatic treatment There is no specific treatment for ricin intoxication, although the signs and symptoms can be treated. Death from ricin intoxication occurs within 36–48 h, independent of the route of exposure. If the victim has not died within 3–5 days, then Fig. 3 – The structure of ricin and abrin. survival is highly likely. Avoiding exposure is the best 68 journal of applied biomedicine 12 (2014) 63–77

‘‘treatment’’. A victim of oral ricin poisoning should be treated endocytosis. The A chain is translocated to the cytosol and by lavage and given syrup of ipecac, cathartics, electrolytes, induces cell death by inhibiting protein synthesis (Olsnes, and (possibly) activated charcoal. If ricin is inhaled, treatments 2004; Tang et al., 2007). may include oxygen, bronchodilators, endotracheal intuba- tion, and supplemental positive end-expiratory pressure, as Potential use as a bioweapon and in biocrime needed (Maman and Yehezkelli, 2005; Musshoff and Madea, To date, abrin has not been used as a military weapon in 2009). terrorist attacks. However, because of its high toxicity, accessibility, and relative ease of preparation, its potential Abrin toxin for use as a biological weapon should not be ignored (Roxas- Duncan and Smith, 2012; CDC, 2013). Origin Similar to ricin, abrin toxin is a plant glycoprotein, which is Symptomatic treatment obtained from the seeds of A. precatorius L. commonly known It is imperative that abrin is removed from the body as quickly as jequirity bean and rosary pea. However, abrin is much more as possible. Abrin poisoning is treated by supportive medical poisonous than ricin. All parts of A. precatorius plant are toxic, care, which aims to minimize its effects. The type of care but the seeds contain the highest concentration of toxins depends on several factors, including the route of exposure. (Bhaskar et al., 2012; Roxas-Duncan and Smith, 2012). The Measures include respiratory support, intravenous fluids, and rosary pea, which is the source of abrin, is common to many the use of medications to treat seizures and low blood tropical areas throughout the world and is sometimes used as pressure. Activated charcoal can be used to remove the poison an herbal remedy. The seeds of the rosary pea have been used from the stomach and affected eyes can be thoroughly rinsed to make beaded jewelry, which can lead to abrin poisoning if with water (CDC, 2013). the seeds are swallowed (CDC, 2013).

Chemical structure Bacterial toxins Abrin exists in two forms, namely, abrin a and abrin b. Both comprise two chains: an A chain and a B chain (Patocka, 2011). Most bacterial toxins are large proteins that affect the nervous The A chain comprises 251 residues and is divided into three system (neurotoxins) or damage membranes. They are folded domains, and the B chain is a galactose-specific lectin generally more difficult to produce on a large scale than plant that facilitates the binding of abrin to cell membranes (Chen toxins, but are much more toxic. et al., 1992; Patocka, 2011). The B chains of abrin a and abrin b are composed of 268 amino acids (256 of which are identical). Botulinum toxins The ricin B chain is 60% homologous to the abrin B chain at the amino acid level and the two saccharide-binding sites within Origin the ricin B chain are highly conserved in the abrin B chain is produced by bacteria of (Kimura et al., 1993). The structure of abrin is shown in Fig. 3. botulinum. C. botulinum is a Gram-positive anaerobic bacterium

that produces 8 serologically different types (A, B, C1,C2,D,E,F, Toxicity and symptoms and G), but only serotypes A and B are commercially available. Abrin is much more poisonous than ricin. Abrin can be Serotype A appears to be the most potent type, while serotype separated into three groups (I, II, and III), all of which show C2 is not (Trindade De Almeida et al., 2011). m different mouse i.p. LD50 values: 22, 2.4, and 10 g/kg body weight, respectively (Hedge et al., 1991). The overall i.p. toxicity Chemical structure is 20 mg/kg body weight (Lin et al., 1969; Garber, 2008). If abrin is Botulinum toxin comprises a heavy chain (100 kDa) and a light inhaled, initial symptoms may occur within 8 h of exposure, chain (50 kDa) joined by a disulfide bond. The light chain is an and if it is ingested, initial symptoms may occur in less than that attacks fusion proteins (SNAP-25, syntaxin or 6 h; however, it is more usual for symptoms to appear after 1–3 synaptobrevin) present at the neuromuscular junction, pre- days. Death from abrin poisoning may occur within 36–72 h, venting vesicles from anchoring to the membrane and depending on the dose and route of exposure (inhalation, releasing acetylcholine (Fig. 4). The toxin itself is rapidly ingestion, or injection). If death has not occurred within 3–5 destroyed by heat (i.e., cooking), whereas the that days, the victim usually recovers (CDC, 2008, 2013). produce the toxin are heat-tolerant and will survive in boiling water for an extended period of time (Licciardello et al., 1967; Transmission Setlowa, 2007). Abrin poisoning is not contagious and cannot be spread from person-to-person through casual contact. However, abrin can Toxicity and symptoms be transmitted through the skin via small pellets or projectiles The human LD50 for inhaled botulinum toxin is 3 ng/kg body designed to carry this toxin (CDC, 2008). weight (Physician Information Link, 2004). Naturally occurring can take one of three forms: food-borne, wound, or Mechanism of action intestinal. After food-borne intoxication there is an asymp- Abrin acts in a manner identical to that of ricin, but is much tomatic phase lasting for approximately 18–36 h. Botulism is more potent. The abrin B chain binds to eukaryotic cells via characterized by descending, symmetric, flaccid muscle cell-surface galactosides. The molecule is then taken up by paralysis that first manifests in the bulbar muscles. The journal of applied biomedicine 12 (2014) 63–77 69 [(Fig._4)TD$IG]

and E cleave synaptosomal-associated protein of 25 kd (SNAP- 25), and type C cleaves syntaxin. Muscles are unable to contract in the absence of acetylcholine (Arnon et al., 2001; Noeller, 2001). When used cosmetically, botulinum toxin (Botox) induces muscle paralysis lasting 3–6 months.

Potential use as bioweapon and in biocrime Botulinum toxin is the most poisonous substance known. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people (Arnon et al., 2001). Moreover, botulinum toxin is easy way to produce, transport, and misuse. Those affected require prolonged intensive care (Arnon et al., 2001). As a weapon, the toxin might be released into the air or hidden in the food supplies.

Symptomatic treatment The treatment for botulism is largely supportive. Early treatment with an antitoxin is essential to limit nerve damage. The most serious complication is respiratory failure; therefore, treatment aims to maintain an adequate oxygen supply. Patients may remain on mechanical ventilation in intensive care units, and recovery occurs when the body produces new – Fig. 4 A schematic representation of the domains of the nerve fibers, which can take anywhere from weeks to months botulinum toxin. The binding domain is represented in light (Noeller, 2001). green, the translocation domain in dark green, and the endopeptidase domain in purple. Source: Sesardic, D., Gaines Das, R., 2009. Botulinum toxin: applying the 3Rs to product potency testing. National Origin Institute for Biological Standards and Control. http://www. Shiga toxin belongs to a large family of plant and bacterial nc3rs.org.uk/news.asp?id=1023. protein toxins. It kills cells by entering the cytosol and inhibiting protein synthesis (Sandvig, 2001). Shiga toxin is produced by dysenteriae and Shiga toxin-producing patients appear lethargic and have difficulty communicating Escherichia coli (STEC). STEC are now recognized as an due to bulbar palsy. Prominent bulbar palsies can be summa- important group of bacterial enteropathogens. There are at rized in terms of the ‘‘four Ds’’: diplopia, dysarthria, dyspho- least 100 serotypes of E. coli that are capable of producing Shiga nia, and dysphagia. Other symptoms may include blurred toxins; the most well-known of these is E. coli O157 (Riley et al., vision, dry mouth, and mydriasis (Arnon et al., 2001; Noeller, 1983; Law, 2000). 2001). Chemical structure Transmission Shiga toxin comprises two subunits, A and B. The Shigella B Botulism is not spread from person-to-person. Transmission subunit comprises a pentameric ring, which encircles a helix can occur through eating foods contaminated with botulinum at the C terminus of a single A subunit. The B subunit binds to spores, which subsequently grow into bacteria that produce [(Fig._5)TD$IG] the botulism toxin. Wound botulism occurs when bacteria (C. botulinum) contaminate a wound, germinate, grow within the wound, and produce toxins that are absorbed directly into the bloodstream. Infant botulism occurs when an infant con- sumes the spores of C. botulinum, which then grow and secrete toxins into the baby's intestines (Noeller, 2001; Galassi et al., 2011).

Mechanism of action Botulinum toxin is a zinc endopeptidase that binds to the neuronal at the nerve terminus. It then enters the neuron by endocytosis. The light chain of botulinum toxin cleaves specific sites within the soluble N-ethylmaleimide- sensitive fusion protein (NSF)-attachment protein receptor, preventing complete assembly of the synaptic fusion complex and irreversibly blocking the release of acetylcholine. Type B, D, F, and G botulinum toxins cleave synaptobrevin, types A, C, Fig. 5 – Crystallographic structure of Shiga toxin. 70 journal of applied biomedicine 12 (2014) 63–77

receptors at the cell surface and the toxin enters the including managing slaughterhouse operations to minimize cell via macropinocytosis. The enzymatically active A frag- of , the pasteurization of milk and dairy ment contains an internal disulfide bond. Proteolytic cleavage products, and careful washing of and (peeling of the loop area and reduction of the internal disulfide bond is recommended if eaten raw) (Division of Community and within the cytosol releases two fragments: A1 and A2. Similar Public Health, 2011). to ricin and abrin, the A1 fragment inhibits protein synthesis by removing one adenosine from the 28S RNA of the 60S ribosomal subunit (see below). The crystallographic structure Marine Toxins of Shiga toxin is shown in Fig. 5 (Paton and Paton, 1998; Sandvig, 2001). Marine toxins are naturally occurring chemicals that can contaminate certain types of seafood. Contaminated seafood Toxicity and symptoms often looks, smells, and tastes normal, but can make people

When injected i.p. into mice, the LD50 of Shiga toxin is 28 ng sick if ingested. Marine toxins can be used as biological (Yutsudo et al., 1986). Humans infected with STEC initially weapons by terrorists, but they are unlikely to be threat in an suffer from watery diarrhea, which can become bloody within open battlefield situation (Franz, 1996). 1 or 2 days due to hemorrhagic (Riley et al., 1983). Severe abdominal pain is also common. In some patients, STEC Anatoxins infection progresses to hemolytic uremic syndrome (HUS), a life-threatening condition characterized by a triad of acute Origin renal failure, microangiopathic hemolytic anemia, and throm- Anatoxins are a group of neurotoxic alkaloids produced by bocytopenia. Some individuals with HUS experience neuro- cyanobacterial genera including Anabaena, Oscillatoria, Plantko- logical symptoms including lethargy, severe headache, thrix, Microcystis, Cylindorspermum, Phormidium, and Aphanizo- convulsions, and encephalopathy. Although HUS occurs in menon (Rodriguez et al., 2006a,b; Patocka et al., 2011). all age groups, it is more common in infants, young children, Cyanobacteria, formally called ‘‘blue-green algae’’, are simple and the elderly (Tesh and O'Brien, 1991; Paton and Paton, 1998). and primitive photosynthetic microorganisms, which widely occur in fresh, brackish and (Patocka et al., 2011). Transmission STEC can potentially enter the human food chain from a Chemical structure number of animal sources, but the most common is meat that Anatoxins are divided into three types: anatoxin-a, homo- has been contaminated with or intestinal contents after anatoxin-a, and anatoxin-a(s) (Fig. 6) (Patocka et al., 2011). slaughter (Paton and Paton, 1998). A major source of exposure Anatoxin-a is a low molecular weight (165 g/mol) bicyclic is contaminated undercooked ground beef, although other secondary amine, 2-acetyl-9-azabicyclo(4-2-1)non-2-ene. Ana- foods such as unpasteurized milk, juice, and contaminated toxin-a in the protonated form is more stable than the free raw fruits and vegetables are also potential sources. Direct base. It has a pKa of 9.4 and exists in the protonated form at contact with animals and their environment is also a risk physiological pH (Duy et al., 2000). This protonated from is factor. Waterborne transmission can occurred through the more stable than the free base. Anatoxin-a is relatively stable consumption of inadequately chlorinated water and by in the dark, but in pure solution in the absence of pigments it swimming in contaminated lakes and pools. The infectious undergoes rapid photochemical degradation upon exposure to dose is very low, and person-to-person transmission is sunlight. Breakdown is accelerated under alkaline conditions common (Division of Community and Public Health, 2011). (Stevens and Krieger, 1991). Homoanatoxin-a (MW = 179 g/mol) is a homologue of Mechanism of action anatoxin-a; it carries a propionyl group at C-2 instead of an Shiga toxins inhibit protein synthesis within target cells via a acetyl group. Anatoxin-a(s) is a unique phosphate of a mechanism similar to that used by ricin. After entering a cell cyclic N-hydroxyguanine (Fig. 4) (Matsunaga et al., 1989; via macropinosomes, the protein functions as an N-glycosi- Sivonen and Jones, 1999), which decomposes rapidly under dase, cleaving a specific adenine nucleobase from the 28S RNA basic solutions but is relatively stable under neutral and of the 60S ribosomal subunit, thereby inhibiting the peptide acidic conditions (Sivonen and Jones, 1999). The compound chain elongation step of protein synthesis and ultimately is inactivated at elevated at temperatures above 40 8C causing cell death (Igarashi et al., 1987; Endo et al., 1988; Paton (Carmichael, 2001). and Paton, 1998). [(Fig._6)TD$IG]

Potential use as a bioweapon and in biocrime To date, there are no reports of this toxin being used as military or terrorist weapons, but its military potential as a toxin weapon is very high.

Symptomatic treatment If contact occurs, the eyes, skin or wounds should be flushed thoroughly with water and medical attention sought as Fig. 6 – Chemical structure of anatoxin-a, homoanatoxin-a, quickly as possible. Basic control measures can limit exposure, and anatoxin-a(s). journal of applied biomedicine 12 (2014) 63–77 71

Toxicity and symptoms ceases. However, it is important to carefully monitor the patient Anatoxin-a causes death within minutes to a few hours, for further anticholinergic effects and to either reduce or depending on the species affected, the amount of toxin discontinue atropine should any adverse effects become ingested, and the amount of food in the stomach. The LD50 apparent (Patocka et al., 2011). Treatment with oxime reacti- for anatoxin-a(s) when injected i.p. into mice is 20 mg kg/body vators and atropine, or pretreatment with a carbamate and weight (Carmichael et al., 1990). The symptoms of anatoxin-a atropine, have been explored as potential treatments for, and poisoning resemble those of nerve agent poisoning and clinical in vivo protection against, respectively, anatoxin-a(s) poisoning. progression is as follows: muscle fasciculations, reduced Anatoxin-a(s) is an active site-directed inhibitor of acetylcho- movement, abdominal breathing, cyanosis, convulsions, and linesterase, which is resistant to oxime reactivation due to the death (Carmichael, 2001). Anatoxin-a causes serious reductions structure of its enzyme adduct. In vivo pretreatment with in blood pressure, heart rate, and gaseous exchange (pO2 and physostigmine and high concentrations of 2-PAM are the only pCO2), resulting in hypoxia, respiratory arrest, severe acidosis, effective treatments for a lethal dose of anatoxin-a(s) (Hyde and and death (Devlin et al., 1977; Adeymo and Siren, 1992). Carmichael, 1991). Initially, anatoxin-a(s) slows the heart rate and reduces arterial blood pressure, and these parameters become worse (STX) with time (Cook et al., 1989). In addition, anatoxin-a(s) induces hypersalivation in mammals, as well as other symptoms more Origin typical of such as diarrhea, shaking, and a nasal Saxitoxin (STX) and its analogs, collectively termed paralytic mucus discharge (Cook et al., 1990). shellfish (PSPs), are highly potent neurotoxins. STX and its analogs are synthesized by dinoflagellates of the genus Transmission Alexandrium, including A. tamarense and A. cantenella, in both The toxin can enter the body by inhalation and injection. marine and freshwater environments (Oshima et al., 1993; When exposed to high concentrations, it can enter the body Lefebvre et al., 2008; Faber, 2012). Several freshwater species of through skin (Patocka and Streda, 2002). filamentous cyanobacteria, such as Anabaena circinalis, Apha- nizomenon sp., Aphanizomenon gracile, Cylindrospermopsis raci- Mechanism of action borskii and Lyngbya wollei, also produce STX (Lagos et al., 1999; Anatoxins act in a manner similar to that of organophospho- Pearson et al., 2010). Blooms of these toxic species can lead to rus and carbamate insecticides (Cook et al., 1988). Anatoxin-a the mass killing of fish, native animals, and livestock, and the and homoanatoxin-a are postsynaptic depolarizing neuro- contamination of freshwater resources (Negri et al., 1995). muscular blocking agents. They mimic the neurotransmitter, acetylcholine, by irreversibly binding to the nicotinic acetyl- Chemical structure choline receptors of the sodium channels within the postsyn- STX belongs to the large family of natural marine products that aptic neurotransmitter plates (Carmichael et al., 1977; Spivak harbor two guanidino groups, which are responsible for its et al., 1980; Patocka and Streda, 2002). This blocks further high polarity (Shimizu, 2000; Berlinck and Kossuga, 2005; electrical transmission; thus a sufficiently high dose paralyzes Wiese et al., 2010). STX and its 57 analogs vary in potency, and the muscles (including the respiratory muscles), leading to are categorized into subgroups based on the presence of death by asphyxiation (van Apeldoorn et al., 2007). substituent side chains comprising carbamates, sulfates, Anatoxin-a(s) is an organic phosphonate and acts in a hydroxyls, hydroxybenzoates, or acetates (Fig. 7) (Faber, manner similar to synthetic organophosphonate nerve agents. 2012). STX is soluble in water, methanol, and ethanol, but However, anatoxin-a(s) acts only in the periphery; cholines- not in other organic solvents, and can bio-accumulate through terase activity in the brain and retina remain normal, even in different trophic levels (Wiese et al., 2010). animals given a lethal dose (Briand et al., 2003). Anatoxin-a(s) binds to acetylcholinesterase, rendering it unable to hydrolyze Toxicity and symptoms acetylcholine. Therefore, acetylcholine is not removed from STX is a powerful neurotoxin. When administered orally or i.p., m m the nicotinic receptor by cleavage into its two component the LD50 for mice is 263 g/kg (Price et al., 1991) and 10 g/kg parts, acetic acid and choline. The ion channel therefore (Landsberg, 2002), respectively. Humans have died after remains open, destroying muscle function through exhaustion ingesting as little as 1 mg of toxin (Evans, 1969). In mice, (Mahmood and Carmichael, 1987; Patocka and Streda, 2002; STX has a lowest-observed-adverse-effect-level (LOAEL) of Dittmann and Wiegand, 2006). 1.5[(Fig._7)TD$IG] mg/kg body weight and a no-observed-adverse-effect-level

Potential use as a bioweapon and in biocrime The potential use of anatoxins as a military weapon is very high. They are thermally labile and are inactivated by heat. The toxins can enter the body by ingestion, injection, inhalation, or through abraded skin. To date, there are no reports of these toxins being used as military or terrorist weapons.

Symptomatic treatment Exposure to sub-lethal doses of anatoxin-a(s) can be treated with atropine, which is repeatedly administered until salivation Fig. 7 – Chemical structure of Saxitoxin. 72 journal of applied biomedicine 12 (2014) 63–77 [(Fig._8)TD$IG]

(NOAEL) of 0.5 mg/kg body weight. Human mortality is variable and has been observed at oral doses of 500–2400 mg (Faber, 2012). STX and its derivatives are the causative agents of a common form of seafood poisoning called PSP, which is life- threatening. Symptom onset is rapid and purely neurological. Within 30 min of ingestion, victims become aware of tingling or numbness around the lips, which gradually spreads to the face and neck. This is followed by a prickly sensation in the Fig. 8 – Chemical structure of tetrodotoxin (TTX). fingertips, headache, fever, nausea, vomiting, and diarrhea. Source: Nieto et al., 2012. The most severe cases result in respiratory arrest due to respiratory muscle paralysis within 24 h of consumption (Daranas et al., 2001; Llewellyn, 2006). Tetrodotoxin (TTX) Transmission Herbivorous zooplankton infected with STX can transmit the Origin toxin to fish and other aquatic creatures. Humans develop PSP Tetrodotoxin (TTX) is a potent neurotoxin that blocks the after ingesting contaminated raw or cooked shellfish that sodium channels within the cell membranes of neurons. It was contain high concentrations of STX (Faber, 2012). first isolated as a crystalline prism from toxic puffer fish by Yokoo (1950). TTX is also found in other marine organisms, Mechanism of action including some species of gobies, octopuses, sea stars, crabs, STX is lipid soluble; therefore, it is readily absorbed via the and gastropods (Hashimoto et al., 1990). TTX is also produced gastrointestinal and respiratory tracts. STX is a potent by bacteria that reside within blue-ringed octopuses (Noguchi neuroparalytic agent that binds to the sodium channels et al., 1986). The most common bacteria associated with TTX within nerve and muscle tissue. It has high affinity for binding production are Vibrio bacteria, with Vibrio alginolyticus being the on site 1 on the voltage-gated sodium ion channel and binds most common species (Hwang et al., 1988). tightly to a cell-surface receptor very close to the external orifice of the sodium channel. In humans, this blocks the influx Chemical structure of sodium ions through the membrane, prevents depolariza- Tetrodotoxin is compound which has a low molecular weight, tion, and inhibits subsequent impulse-generation in the non protein based small molecule with a unique cage structure peripheral nerves and skeletal muscles, leading to paralysis (Fig. 8). Its three nitrogen atoms make a positively charged (Kao, 1972, 1983; Narahashi, 1972; Faber, 2012). This occurs guanidinium group. Hydroxyl groups on the fused ring because the calcium voltage-gated channels do not open and systems increase the ability to react in aqueous environment. potassium and chloride conductance remains unaltered This toxin is odorless and heat stable but unstable at pH levels (Faber, 2012). above 8.5 and below 3.0 (Mirbakhsh, 2004).

Potential use as a bioweapon and in biocrime Toxicity and symptoms STX is classified as Agent TZ (chemical weapons designation) TTX is among the most toxic substances known to man. It has and is considered to have potential for use in chemical a lethal potency of 5000–6000 MU/mg, and the minimum lethal weapons due to its high toxicity. STX dissolves easily in water dose (MLD) for humans is approximately 10,000 MU (2 mg) and can be ingested or inhaled. Studies show that it is possible (Noguchi and Ebesu, 2001). to spike ammunition with STX, thereby ensuring the immedi- The first symptoms occur 15 min to several hours after ate death of the victim. STX is much more poisonous than the ingesting TTX-contaminated food. Initial symptoms include synthetic nerve gas sarin; therefore, like ricin, it is classified as lip and tongue paresthesia, followed by facial and extremity a biological weapon (Federal Department of Defense, Civil paresthesia and numbness. Salivation, nausea, vomiting, and Protection and Sports (DDPS), 2012). However, because it diarrhea with abdominal pain develop quickly. Motor dys- cannot be chemically synthesized efficiently or easily pro- function, muscle weakness, hypoventilation, and speech duced in large quantities from natural sources, it is unlikely to difficulties soon follow. A rapid ascending paralysis occurs be used as an aerosol weapon on the battlefield. over the course of 4–24 h. Extremity paralysis precedes bulbar paralysis, which is followed by respiratory muscle paralysis. At Symptomatic treatment first, deep tendon reflexes are preserved. Finally, cardiac There is no antidote to STX. Supportive therapy is the rule and dysfunction with and dysrhythmias (bradycar- survivors usually show a full recovery (Daranas et al., 2001). dia), dysfunction (e.g., coma), and Fortunately, those exposed to low levels of STX can be treated seizures develop. Patients experiencing severe toxicity may with measures such as Benzedrine (to enlarge the airways) and enter a deep coma, with fixed non-reactive pupils, apnea, and gastric lavage with activated charcoal or a dilute bicarbonate loss of all brain stem reflexes. TXT is equally dangerous solution (Kao, 1983). STX-related mortality depends on the regardless of whether it is ingested, inhaled, absorbed through time elapsed before the victim receives medical care, including the skin, or enters the bloodstream in any other way. Typically, fluid therapy, assisted ventilation, and blood pH monitoring death occurs from respiratory muscle paralysis and respirato- (Kao, 1983). There is no human vaccine against STX. ry failure (Benzer, 2001). journal of applied biomedicine 12 (2014) 63–77 73

Transmission the genus , which are found in the rain forests of No person-to-person transmission is reported. TTX may be western . Three species of true poison-dart frogs exposed via skin contact, skin absorption, eye contact, secrete atrachotoxins: Phyllobates terribilis, , inhalation, and ingestion. and . The main alkaloids that make up the toxins are atrachotoxin, homobatrachotoxin, and batracho- Mechanism of action toxinin-A (Fig. 9) (Myers et al., 1978; Daly et al., 2005). BTX has TTX is much larger than a sodium ion and therefore acts like a also been extracted from the feathers of birds (, Ifrita cork in a bottle. The toxin binds to the neurotoxin receptor site 1 kowaldi, and Colluricincla megarhyncha). It is the most potent on the a-subunit within the outer vestibule of voltage-gated non-peptide neurotoxin known (Dumbacher et al., 1992, 2000). sodium channels and blocks the influx of sodium ions by occluding the outer pore (Fozzard and Lipkind, 2010). TTX is Chemical structure specificfortheNa+ ion channel; therefore, it blocks the flow of Na+ BTX was named by scientists John Daly and Bernard Witkop, ions while having no effect on that of K+ ions. Binding to the who isolated the pure and determined its structure channel is relatively tight. Whereas hydrated sodium ions bind and chemical properties. BTX is the (20-alpha)-2,4-dimethyl- reversibly on a nanosecond time-scale, TTX is bound for tens of pyrrole-3-carboxylate of batrachotoxinin-A (Daly, 1998). The seconds. This inhibits the propagation of action potentials, structure of BTX includes a steroid skeleton and an oxazapane paralyzes nerve and muscle function, and causes death by induc- ring (Kurosu et al., 1998) (Fig. 9). ing respiratory failure (Lee and Ruben, 2008; Mirbakhsh, 2004). Toxicity and symptoms Potential use as a bioweapon and in biocrime BTX is a strong cardiotoxin, which affects ion permeability There is no report of using TTX as a bioweapon, but due to its leading to an irreversible depolarization of nerves and muscles high toxicity, its potential as a weapon of terror cannot be resulting in arrhythmias, fibrillation, and cardiac failure discounted. (Albuquerque et al., 1971; Bosmans et al., 2004). Symptoms in laboratory animals include strong muscle contractions, Symptomatic treatment violent convulsions, salivation, dyspnea and death (even at There are no clinically approved antidotes for TTX (Benzer, doses of <0.1 mg) (Patocka et al., 1999). The leading cause of 2001). However, anti-TTX monoclonal antibodies proved to be death from BTX is usually from cardiac failure. a life-saving treatment in mouse models both before and after ingestion of a lethal dose (Mirbakhsh, 2004). Transmission No person-to-person transmission of BTX is reported.

Venom toxins Mechanism of action BTX are potent modulators of voltage-gated sodium channels, Many known venom toxins have been sequenced, and some which orchestrate the generation of action potentials and are have been cloned and expressed. Some small venom toxins the key elements in signal in neurons and other have been generated using relatively simple chemical synthe- excitable cells (Wang and Wang, 1994; Catterall, 2000). BTX sis techniques. However, they are difficult to produce in large binds to the sodium channels and shifts the threshold voltage quantities for use in bioweapons and their threat potential is to a more negative value, which causes an irreversible change limited (Franz, 1996). in the channel's conformation, so it is permanently open (Li et al., 2002). Studies show that BTX binds so tightly that even (BTX) washing for 3 h had no effect (Albuquerque et al., 1971). The permanently open channel allows the free passage of ions, Origin resulting in the loss of the Na+/K+ concentration gradient; thus Batrachotoxins (BTX) are extremely potent cardiotoxic and nerve and muscle cells can no longer transmit electrical [(Fig._9)TD$IG]neurotoxic steroidal alkaloids secreted by poison-dart frogs of signals (Wang et al., 2006).

Fig. 9 – Chemical structure of batrachotoxins. 74 journal of applied biomedicine 12 (2014) 63–77

Potential use as a bioweapon and in biocrime Anderson, P.D., 2012. Bioterrorism: toxins as weapons. J. Pharm. m Pract. 25, 121–129. The subcutaneous LD50 for BTX in mice is 0.2 g/kg, and doses Arnon, S.S., Schechter, R., Inglesby, T.V., Henderson, D.A., as low as 0.01 and 0.02 mg/kg can be lethal (Jiri and Stredab, Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Hauer, J., 2002). There is no evidence to suggest that BTX has been used Layton, M., Lillibridge, S., Osterholm, M.T., O'Toole, T., by the military or by terrorists as a biological weapon. The Parker, G., Perl, T.M., Russell, P.K., Swerdlow, D.L., Tonat, K., toxin is most effective when injected using a dart or needle; 2001. Botulinum toxin as a biological weapon-medical and however, dipping the dart/needle in botulinum toxin would be public health management. JAMA 285, 1059–1070. much more effective (Patocka et al., 1999). Audi, J., Belson, M., Patel, M., Schier, J., Osterloh, J., 2005. 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