FINAL Ricin EPA San Diego.pdf

Ricin:
A phytotoxin from the castor bean
(Ricinus communis)
Dr. Mark A. Poli, PhD, DABT
US Army Medical Research Institute of Infectious
Diseases, Fort Detrick, Maryland

Ricin is a Plant Toxin

From: Sarah L. Corbett, Valdosta State University
Castor Beans

Castor Beans are an Important
Agricultural Commodity
• Commercial uses
• Paints, dyes, varnishes
• Lubricant
• Purgative/laxative
• Source of sebacic acid, used
in the production of nylon
• Animal food supplement
• Fertilizer
From: Thai Castor Oil Industries

But Inside the Beans . . .
(267 a.a.)
• MW approx 66 Kd
• Inhibitor of protein synthesis
• Depurination of A4324 disrupts
binding of elongation factor 2
(EF2) leading to termination of
protein synthesis
(262 a.a.)
Model courtesy of Dr. Mark Olsen, USAMRIID

How it Works . . .
• B chain is lectin, which directs binding to galactose residues
on cell surface proteins
• Endocytic uptake is primarily (but not exclusively) through
uncoated pits
• Once in endosomes, either
S recycled to cell surface
S degraded in lysosomes
S enter the cytosol via the Golgi apparatus
• Separation of A and B chains
• Enzymatic cleavage of specific adenosine residue from 28S
ribosomal subunit, which inhibits binding of EF-2

Clinical Features . . .
Ingestion:
• dose-dependent dependent latency period (up to 10 hr)
• fever, thirst, sore throat, headache
• nausea, vomiting, abdominal cramps
• severe diarrhea, GI hemorrhage, vascular collapse
• necrosis of the liver, spleen, kidneys, lymph nodes
• death in 3-53
5 days
`

Clinical Features . . .
Inhalation:
• dose-dependent dependent latency period (8 – 24 hr)
• fever, nausea, tightness in the chest
• cough, dyspnea
• hypothermia and cyanosis secondary to massive
pulmonary edema
• upper airway necrosis
• death in 36-48 hr (high dose)

Pulmonary Exposure

Medical Management is Limited
to Supportive Care . . .
For inhalation exposure:
S treat the acute lung injury and pulmonary edema
For ingestion:
S gastric lavage, , cathartics
S i.v. . fluids and electrolyte replacement

Prophylaxis . . .
• Physical protection
S respiratory protection (mask)
S not dermally active
• No vaccine available for human use
S vaccine candidates scheduled for
advanced development

Diagnostics . . .
Typically direct capture immunoassays
Hand held assays are available
S New Horizons Diagnostics, Columbia, MD
S Tetracore, Gaithersburg, MD
S Jant Pharmacal, Encino, CA
Activity assays
Immunohistochemistry

Direct Capture Immunoassay
biotinylated antibody
ricin
ruthenylated antibody
streptavidin-coated paramagnetic bead

Flow Cell
photodiode
electrode
magnet
Magnetic beads

Clinical Matrices Are Not a Problem
7000
10% Human Serum
4000
Urine
6000
Relative ECL Units
5000
4000
3000
2000
1000
Relative ECL Units
3000
2000
1000
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
Ricin (ng/ml)
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Ricin Conc (ng/ml)

Activity Assay
5’
RNA substrate
A G C G G G A G A
3’ C C C U C U
G
A
dA
G
Free toxin
(pH= 4.1)
Deadenylation site
Deadenylated
product
5’
*
Me 2 ED, beads, Ru-ODN
Detect
RuODN anneals to
unblocked product
5’-Ru

ECL-Based Ricin Activity Assay
8
Signal/Background
7
6
5
4
3
2
1
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
Ricin (ng/ml)

Immunohistological Detection
in Tissue Samples
Courtesy of Dr. Chad Roy, USAMRIID

Harnessing Cytotoxicity for Medical
Advantage: Immunotoxins
• Cytotoxic activity can be targeted
to individual cell types by covalently
linking toxins to antibodies specific
for cell surface receptors
• Approach is limited by side effects:
S nonspecific hepatic cytotoxicity
S vascular leak syndrome
• Currently undergoing phase 1
clinical trials for the treatment of
lymphoma, myeloma, and graft-vs.-
host disease

Side Effects Have Limited the
Use of Immunotoxins as
Cancer Treatment . . .
• Nonspecific hepatotoxicity
S 1980s: Minimized by deglycosylation – stripping of sugar residues
• Vascular Leak Syndrome
S determined to a separate event from cytotoxicity, involving a
different portion of the protein sequence
S single AA substitution eliminated the VLS without affecting
cytotoxicity
S LD 50 in mice increased 5-fold5
S 5x higher dose improved therapeutic value in mouse model (survival
time)
(Smallshaw,, J. et al. (2003) Nature Biotechnology 21:387-391
391)

Ricin and The Terrorist

Listed by the CDC as a
Category B Agent . . .
• Moderately easy to disseminate
• Moderate morbidity rates/low mortality rates
• Requires specific enhancements in diagnostic
capacity

Why Ricin?
• Extremely potent by aerosol route
S LD 50 in animal models 3-53
µg/kg
• Widely available in large quantities
S grown in third-world nations
• Easily extracted with low technology

A Little Recent History . . .
• 1995: agents discover 130 g of ricin at the Canadian border
• 2002: six terrorist suspects arrested in Manchester, England
• 2003: subtoxic quantities of ricin discovered in the Paris Metro;
thought to be related to a plan to attack the Russian embassy
• 2003: envelope with a threatening note and a sealed container of o
ricin processed at a mail facility in Greenville SC. Note threatened tened to
poison water supplies
• 2004: traces of ricin discovered in the mail room of the Dirksen
Senate Office Building
None of these events resulted in human intoxication . . .
but the toxin is clearly out there!

Possible Mass Casualty
Scenarios . . .
• Distribution into the food or water
supply
• Aerosol dissemination

Distribution in Food or Water . . .
Not a particularly scary scenario
• Poorly absorbed from the GI tract
• LD 50 about 20 mg/kg (mice)
• Death is rare in human intoxications – 2 since
1930

Distribution in Food or Water . . .
• Mass casualty attempt would likely require multi-kg
quantities of pure toxin
• More likely to result in mild intoxication:
S nausea
S vomiting
S diarrhea
• Difficult to differentiate from other foodborne illness

Aerosol Dissemination . . .
The bad news: definitely the worst scenario
• Very toxic by this route – LD 50 3-5 µg/kg*
LD 50
• Particles

Aerosol Dissemination . . .
The good news: it’s s difficult to accomplish
• Efficient aerosol distribution of particles in the
correct size range is very difficult for the non-expert
• Particle size depends upon a variety of factors,
many of them environmental
• Aerosol toxicity is highly dependent upon particle
size

Particle Size is Critical
120
MMAD = 1.0 µm
GSD = 1.3
100
Dose (ng)
80
60
MMAD = 5.0 µ m
GSD = 1.9
MMAD = 12.0 µm
GSD = 1.1
Collison nebulizer vs.
Spinning Top Aerosol Generator
40
20
0
0.1 1.0 10.0 100.0
Aerodynamic diameter (µm)
Roy, C.J, M. Hale, J.M. Hartings, and L. Pitt (2003) Inhalation Toxicology 15:619-638

In the Event of a Mass
Casualty Event . . .
Look for epidemiological clues pointing to a toxin
• Rapid onset of symptoms
• Unusual clusters of cases
• Unexplained deaths among the young or healthy
• Unexplained death of animals or fish
Couple this with
• Laboratory confirmation
• A credible threat

Decontamination . . .
Gross decontamination of patients at the
scene
• Soap and water
• Ricin isn’t dermally active Minimal risk of
secondary aerosols
• Risk to health care provider is minimal
Personal protective gear/equipment
• Decon with 0.1% bleach for 15 min

Summary
• Ricin is a plant toxin that is widely available and has
a history with terrorist groups/individuals
• Potency considerations make aerosol dissemination
the only real mass-casualty scenario
• Potency by aerosol is highly dependent upon
equipment and environmental conditions
• Should exposure occur, most morbidity will likely be
mild to moderate
• Standard decon; ; minimal risk to responders

Thank you!!