BW-ch15.pdf

Ricin

Chapter 15

RICIN

MARK A. POLI, P h D * ; CHAD ROY, P h D † ; KERMIT D. HUEBNER, MD, FACEP ‡ ; DAVID R. FRANZ, DVM, P h D § ; and

NANCY K. JAAX, DVM ¥

INTRODUCTION

HISTORY

DESCRIPTION OF THE AGENT

Toxicity

Pathogenesis

CLINICAL SYMPTOMS, SIGNS, AND PATHOLOGY

Oral Intoxication

Injection

Inhalation

Cause of Death

DIAGNOSIS

MEDICAL MANAGEMENT

Vaccination and Passive Protection

Supportive and Specific Therapy

SUMMARY

* Research Chemist, Department of Cell Biology and Biochemistry, Division of Integrated Toxicology, US Army Medical Research Institute of Infectious

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

† Principal Investigator, Center for Aerobiological Sciences, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland 21702

‡ Major, Medical Corps, US Army; Chief, Education and Training, Operational Medicine Department, Division of Medicine, US Army Medical Research

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

§ Colonel, US Army Veterinary Corps (Ret); Vice President and Chief Biological Scientist, Midwest Research Institute, 365 West Patrick Street, Suite

223, Frederick, Maryland 21701; formerly, Commander, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland

¥ Colonel, US Army Veterinary Corps (Ret); Special Projects Officer, National Agricultural Biosecurity Center, 203 Fairchild Hall, Kansas State Uni-

versity, Manhattan, Kansas 66506; formerly, Chief, Division of Pathology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter

Street, Fort Detrick, Maryland

323

Medical Aspects of Biological Warfare

INTRODUCTION

Ricin is a protein isolated from the seeds of the cas-

tor bean plant ( Ricinus communis ). Like abrin (from

the seeds of the rosary pea, Abrus precatorius ), ricin is a

lectin and a member of a group of ribosome-inactivat-

ing proteins that block protein synthesis in eukaryotic

ribosomes. 1

The castor bean is native to Africa, but it has been

introduced and cultivated throughout the tropical

and subtropical world. Although tolerant to a wide

temperature range, it grows best in elevated year-

round temperatures and rapidly succumbs to sub-

freezing temperatures. However, it is often grown as

an ornamental annual in temperate zones. The seeds

are commercially cultivated in many regions of the

world, predominantly in Brazil, Ecuador, Ethiopia,

Haiti, India, and Thailand. The beans contain 35% to

55% by weight of fast-drying, nonyellowing oil used

in the manufacture of lubricants, inks, varnishes, and

dyes. After oil extraction, 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. 2 Although consid-

erably less potent than botulinum neurotoxins and

staphylococcal enterotoxins, ricin represents a signifi-

cant potential biological weapon because of its stability

and worldwide availability as a by-product of castor

oil production. In addition, it has been associated

with several terrorist actions and therefore may be a

potential agent of bioterror.

HISTORY

R communis was cultivated for centuries in ancient

Egypt and Greece for the lubricating and laxative

effects of its oil. In addition, both the oil and whole

seeds have been used in various parts of the world for

disease treatment as well as for malicious mischief and

homicidal purposes. 3 During World War I, the excellent

lubricating properties of castor oil were utilized by the

wartime aircraft industry. Shortages of castor oil dur-

ing World War II resulted in US government subsidies

for agricultural production of castor beans in the San

Joaquin Valley of California. These subsidies persisted

until the 1960s, when synthetic oils replaced castor oil

in the aircraft industry. There is no commercial produc-

tion of castor oil in the United States today.

The first toxinology work on ricin was performed by

Hermann Stillmark at the Dorpat University in Estonia

for his 1888 thesis. 4 Stillmark determined that ricin was

a protein and suggested the name. He purified ricin to

a very high degree (although not completely to homo-

geneity) and found that it agglutinated erythrocytes

and precipitated serum proteins. 5 For years, these ef-

fects were considered to be the mechanism of action of

ricin, although later work showed that the toxicity and

agglutination effects were separable properties.

In 1891 Paul Ehrlich studied ricin and abrin in

pioneering research that is now recognized as the

foundation of immunology. 5 Following the lead of In-

dian farmers who had known for centuries that calves

could be protected from abrin poisoning by feeding

them small amounts of Abrus seeds, Ehrlich vaccinated

animals with small oral doses of castor beans. After

protection was established, he continued vaccinating

with subcutaneous injections of toxin. Experiments

with the serum of immune animals led him to dis-

cover that the immunity was specific, was associated

with serum proteins, and could be transferred to the

offspring through milk.

At the end of the 19th century, with the rising inter-

est in bacterial toxins, interest in plant toxins waned. It

wasn’t until the mid-20th century, with the discovery

that ricin inhibited protein synthesis and thus might

be useful for treating cancer, that the scientific com-

munity “rediscovered” ricin. Olsnes and Pihl 6 demon-

strated that protein synthesis was strongly inhibited

in a cell-free rabbit reticulocyte system, and suggested

that the effects resulted from inhibited elongation of

the nascent polypeptide chain. They also determined

that ricin consisted of two dissimilar polypeptide

subunits and that the A chain was responsible for the

toxic action. Results from this laboratory over the next

few years revealed the 60S ribosomal subunit as the

enzymatic target and led to further characterization of

the enzymatic action. 7

More recently, the inhibitory action of ricin on pro-

tein synthesis in eukaryotic cells was investigated as a

potential chemotherapeutic agent against some forms

of cancer. The active subunit of ricin is specifically

targeted to tumor cells by conjugation to tumor-spe-

cific antibodies. These chimeric toxins, called immu-

notoxins, have been tested against several forms of

cancer, with promising results. 8 However, side effects

such as nonspecific hepatic toxicity and vascular leak

syndrome (VLS) have been problematic and dose

limiting. Recent work by Smallshaw and coworkers 9

has demonstrated that the VLS activity of the toxin

is mediated by a discrete sequence moiety separate

324

Ricin

from the region related to protein synthesis inhibi-

tion. Specifically, mutations in a three-amino acid

motif of the ricin A chain yielded an immunotoxin

with significantly reduced VLS side effects with no

loss of cytotoxicity. Testing in a mouse model demon-

strated improved effectiveness, suggesting that ricin

immunotoxins may yet have a place in the anticancer

armamentarium.

Because of its potency, worldwide availability, and

ease of production, the US Chemical Warfare Service

began considering ricin as a potential biological war-

fare agent near the end of World War I. The research

involved methods of adhering ricin to shrapnel and

the production of effective aerosol clouds. 10 However,

the war ended before the evolution of weaponry based

upon this research. During World War II, the Ameri-

cans and British collaborated on the development of

a ricin-containing bomb (the so-called “W bomb”).

Although they were tested, these bombs were never

used in battle. The United States unilaterally ended

its offensive biological warfare program in 1969–1970;

all offensive research and development were termi-

nated, and remaining stocks of ricin munitions were

destroyed in 1971–1972. The 1975 Convention on

the Prohibition of the Development, Production and

Stockpiling of Bacteriological (Biological) and Toxin

Weapons and on Their Destruction prohibited the

development, production, and storage of any toxin

for offensive purposes.

In addition to its coverage under the 1975 conven-

tion, ricin and one other toxin (saxitoxin) were also

specifically included under the 1993 Chemical Weap-

ons Convention, ratified by Congress in 1997. In the

United States, ricin and abrin are both included in the

Centers for Disease Control and Prevention’s select

agent list of toxins requiring certification for possession

and transfer. The US intelligence community believes

that ricin was included in the biological warfare pro-

grams of the Soviet Union, Iraq, and possibly other

nations as well.

In recent years, ricin has drawn the interest of ex-

tremist groups. Such notoriety is likely driven by the

ready availability of castor beans, ease of toxin extrac-

tion, coverage in the popular press, and popularization

on the Internet. Several individuals have been arrested

under the 1989 Biological Weapons Anti-Terrorism

Act for possessing ricin. In the past few years alone,

various major news organizations have reported the

following stories:

• 2002: Ricin was discovered in the apartment of

six terrorist suspects arrested in Manchester,

England.

• 2003: An envelope containing a sealed con-

tainer of ricin and a note threatening to con-

taminate water supplies was processed at a

mail facility in Greenville, South Carolina.

• 2004: Traces of ricin were discovered in the

mail room of the Dirksen Senate Office Build-

ing in Washington, DC.

While none of these events resulted in any known

human intoxications, they clearly demonstrate that

ricin is well known, available to and recognized by

extremist groups, and should be seriously considered

as a potential bioterrorist threat agent.

DESCRIPTION OF THE AGENT

Ricin is a 66-kd globular protein that typically makes

up 1% to 5% of the dry weight of the castor bean,

although the yield can be highly variable. 11 The toxic

form is a heterodimer consisting of a 32-kd A chain con-

nected to the 32-kd B chain through a single disulfide

bond. 12 As such, it is a member of the type II family of

ribosome-inactivating proteins (RIPs), which possess

enhanced in-vivo toxicity because of the presence of the

B chain that facilitates uptake by the cell. Type I RIPs lack

the B chain, and cellular toxicity is much less; uptake

depends on endocytosis. Both chains are glycoproteins

containing multiple mannose residues on their surfaces;

association of both chains is required for toxicity.

Purification and characterization is not difficult,

and the crystal structure has been determined to .25

nm. 13 Each chain is a globular protein, with the A chain

tucked into a gap between two roughly spherical do-

mains of the B chain. A lactose disaccharide moiety is

bound to each of these spherical domains. The disul-

fide bond links residue 259 of the A chain with residue

4 of the B chain. The crystal structure demonstrates a

putative active cleft in the A chain, which is believed

to be the site of enzymatic action. A functional lipase

active site at the interface of the two subunits was re-

cently identified. 14 This site is thought to be important

for intracellular A chain translocation and subsequent

intracellular trafficking (see below). Recombinant A

and B chains, as well as specific mutants, have been

expressed and characterized in several expression

systems including Escherichia coli . 15-18

Toxicity

Ricin is recognized as one of the most exquisitely

toxic plant-derived RIPs identified to date. 19 However,

considerable variation in potency exists among species.

325

Medical Aspects of Biological Warfare

For instance, on a mg/kg basis, potency varies over

two orders of magnitude between species of domestic

and laboratory animals; chickens and frogs are the least

sensitive, and horses are the most sensitive. 20 Potency

also varies greatly with route of administration. In

laboratory mice, approximate median lethal dose val-

ues and time to death are, respectively, 5 µg/kg and

90 hours by intravenous injection, 22 µg/kg and 100

hours by intraperitoneal injection, and 24 µg/kg and

100 hours by subcutaneous injection. Ricin is extremely

toxic by inhalation; median lethal dose estimates range

from 3 to 15 µg/kg in rodents and primates (Table 15-

1). In contrast, ricin is least potent by the oral route;

median lethal dose estimates in mice are approximately

20 mg/kg. Low potency by the oral route likely reflects

poor absorption and possibly partial degradation in

the gut. Higher potency by other routes may be related

to the ubiquitous nature of toxin receptors among cell

types. In skin tests on mice, no dermal toxicity was

observed at 50 µg/spot, suggesting poor dermal ab-

sorption of this large, highly charged protein. 21

A B

lysosome

degrade

recycle

Golgi apparatus

A B

-KDEL

Calreticulin/others?

Endoplasmic Reticulum

A B

PDI?

refolding

catalysis

Pathogenesis

Protease

degradation

Ribosome

The mechanism of action of ricin is similar to that

of other type II RIPs. The two-chain structure is key to

cellular internalization and subsequent toxicity. The

lectin properties of the B chain enable toxin binding to

cell-surface carbohydrates, and the A chain possesses

the enzymatic activity. Initial binding of the B chain

to glycoside residues on glycoproteins and glycolip-

failure of

EL-2 to bind

Ribose

4324

Fig. 15-1. Binding, internalization, and intracellular track-

ing of ricin leading to enzymatic action at the 60S ribosome.

Endosomes transport ricin from the initial binding site to the

Golgi apparatus (and may also traffic the internalized ricin

back to the cell surface or to lysosomal degradation). Then,

calreticulin and possibly other proteins are thought to chap-

erone the ricin from the Golgi apparatus to the endoplasmic

reticulum (ER). At the ER, protein disulfide isomerase may

reduce the disulfide bridge between the ricin subunits, fa-

cilitating unfolding and retrograde transport of the A chain

through the ER lumen via a Sec61-mediated translocon. In

the cytoplasm, the A chain can interact with the ribosome,

which acts as a suicidal chaperone stimulating proper refold-

ing and resumption of catalytic activity. The A chain cleaves

one specific adenosine residue (A4324) near the 3’ end of 28S

ribosomal RNA, which blocks elongation factor-2 binding,

thus inhibiting protein synthesis.

A: ricin A chain

A4324: adenosine residue 4324

B: ricin B chain

EL-2: elongation factor 2

-KDEL: amino acid sequence at the C-terminal of a soluble

protein in the lumen of a membrane or a C-terminal Lys-

Asp-Glu-Leu sequence

PDI: protein disulfide isomerase

Illustration: Courtesy of Chad Roy, Tulane National Primate

Research Center, Covington, Louisiana.

TABLE 15-1

MEDIAN LETHAL DOSES FOR AEROSOLIZED

RICIN IN VARIOUS ANIMAL SPECIES

Species

Strain

LD 50 ( µ g/kg)

Mouse

BALB/c

11.2

( Mus musculus )

BXSB

2.8

Swiss Webster

4.9

CBA/J, C57/BL/6J,

L2H/HeJ

5.3

A/J

8.2

C3H/HeN

9.0

Rat

Fisher 344

5.3

( Rattus norvegicus )

African green monkey

( Chlorocebus aethiops )

5.8

Rhesus monkey

15.0

(Macaca mulatta)

LD 50 : medial lethal dose

326

Ricin

ids triggers endocytic uptake of the toxin. Increased

binding is observed in cell types rich in mannose

receptors; dissociation of ricin from its binding

sites is increased in the presence of lactose. 22 There

are a number of possible endocytic mechanisms for

cell entry, some of which are independent of cell

coat-binding protein (clathrin) action. 23 Trafficking

of the toxin within the cell from the initial binding

site to the Golgi apparatus occurs via endosomal

transport and is seemingly regulated by intracellu-

lar calcium. 24 Early endosomes may also traffic the

internalized ricin back to the cell surface or to lyso-

somal degradation (Figure 15-1). A Golgi-associated

type II-α protein kinase also largely regulates toxin

transport in specific cell types such as lymphocytes. 25

Association with the Golgi apparatus seems to be

a requirement for further trafficking to the endo-

plasmic reticulum (ER). 26 Transport from the Golgi

apparatus to the ER is thought to be in association

with one or more chaperone proteins, most notably

calreticulin. 27 Once delivered to the ER, protein

disulfide isomerase may reduce the disulfide bridge

between the subunits, facilitating unfolding and retro-

grade transport of the A chain through the ER lumen

via a Sec61-mediated translocon. 28 ER processing and

transport to the cytosol is a critical step; only when

the holotoxin is reduced by novel chaperones such as

protein disulfide isomerase can subsequent ribosomal

inactivation take place in the cytosol. As with related

toxins, transport to the cytosol is the rate-limiting step

during the decline in protein synthesis. 29 Once trans-

ported from the ER to the cytoplasm, the A chain can

interact with the ribosome, which acts as a suicidal

chaperone stimulating proper refolding and resump-

tion of catalytic activity. 28 The Michaelis constant for

enzymatic action at the ribosome is 0.1 µmol/L and

the enzymatic constant is 1,500/min. It cleaves one

specific adenosine residue (A4324) near the 3’ end of

28S ribosomal RNA. This targeted cleavage blocks

elongation factor-2 binding, thus inhibiting protein

synthesis. 30 The rate of ribosomal inactivation easily

overwhelms repair mechanisms and kills the cell.

CLINICAL SYMPTOMS, SIGNS, AND PATHOLOGY

Animal studies indicate that clinical signs and path-

ological changes in ricin intoxication are largely route

specific. Ingestion causes gastrointestinal symptoms

including hemorrhage and necrosis of liver, spleen,

and kidneys; intramuscular intoxication causes severe

localized pain, muscle and regional lymph node ne-

crosis, and moderate systemic symptoms; inhalation

results in respiratory distress with airway and pul-

monary lesions. Transient leukocytosis appears to be

a constant feature in humans, whether intoxication is

by injection or oral ingestion. Leukocyte counts 2- to

5-fold above normal are characteristic findings among

cancer patients receiving ricin immunotoxin therapy,

and also in the case of the Bulgarian dissident Georgi

Markov during his agonizing death after a successful

assassination attempt. 31

few fatal case reports from abroad. A review of the

American Association of Poison Control Center’s Toxic

Exposure Surveillance System from 1983 to 2002 notes

no reported fatalities from ricin poisoning. 33

A recent review article 34 summarizes symptoms of

substantial castor bean ingestion. The authors note

oropharyngeal irritation, vomiting, abdominal pain,

and diarrhea beginning within a few hours of inges-

tion. Local necrosis in the gastrointestinal tract may

lead to hematemesis, hematochezia, and/or melena.

The resultant loss of fluid and electrolytes may lead to

hypotension, tachycardia, dehydration, and cyanosis.

Significant fluid loss may lead to renal failure and

hypovolemic shock. A portion of the toxin is absorbed

through the gastrointestinal tract leading to systemic

signs. In oral (and parenteral) intoxication, cells in

the reticuloendothelial system, such as Kupffer cells

and macrophages, are particularly susceptible, due to

the mannose receptor present in macrophages. 35 The

Oral Intoxication

The

effect on these cells may lead to liver damage, which

may persist for several days and may progress to liver

failure at higher doses.

In 1985 Rauber and Heard 2 summarized the findings

from their study of 751 cases of castor bean ingestion.

There were 14 fatalities in this study, constituting a

death rate of 1.9%—much lower than traditionally

believed. Twelve of the 14 cases resulting in death oc-

curred before 1930. Even with little or no effective sup-

portive care, the death rate in symptomatic patients has

been low—in the range of 6%. The reported number

Ricin is less toxic by oral ingestion than by other

routes, probably due to poor absorption of the toxin

and possibly partial enzymatic degradation in the di-

gestive tract. In animal models, a significant amount of

orally administered ricin is found in the large intestine

24 hours postingestion with limited systemic uptake. 32

Most cases of oral ingestion are related to ingestion of

castor beans, and the severity of intoxication varies

with the degree of mastication of the beans. Review

of the literature reveals mostly nonfatal case reports

of castor bean ingestion in the United States and a

327

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