BW-ch17.pdf

Additional Toxins of Clinical Concern

Chapter 17

ADDITIONAL TOXINS OF CLINICAL

CONCERN

KERMIT D. HUEBNER, MD, FACEP * ; ROBERT W. WANNEMACHER, J r , P h D † ; BRADLEY G. STILES, P h D ‡ ;

MICHEL R. POPOFF, P h D, DVM § ; and MARK A. POLI, P h D ¥

INTRODUCTION

TRICHOTHECENE MYCOTOXINS

History

Description of the Toxin

Mechanism of Action

Clinical Signs and Symptoms of Intoxication

Diagnosis

Medical Management

MARINE ALGAL TOXINS

History

Paralytic Shellfish Poisoning

Neurotoxic Shellfish Poisoning

Amnesic Shellfish Poisoning

CLOSTRIDIAL TOXINS

History

Description of the Epsilon Toxin

Mechanism of Action

Clinical Signs and Symptoms

Medical Management

SUMMARY

* Major, Medical Corps, US Army; Chief, Education and Training, Department of Operational Medicine, US Army Medical Research Institute of Infec-

tious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

† Consultant, Department of Integrated Toxicology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

21702; formerly, Research Chemist, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

‡ Research Microbiologist, Division of Integrated Toxicology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland 21702

§ Section Chief, Anaerobie Bacteriology and Toxins Unit, CNR Anaerobies et Botulisme, Unite Bacteries Anaerobies et Toxines, Institut Pasteur, 28 rue

du Dr Roux, 75724 Paris, France

¥ 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

355

Medical Aspects of Biological Warfare

INTRODUCTION

Several toxins produced naturally by microorgan-

isms and plants are potent, stable, and capable of caus-

ing a wide range of effects leading to incapacitation

or death. These agents can be ingested, administered

percutaneously, or potentially delivered as aerosols at

the tactical level. Although these toxins may be lethal,

the amount of toxin available from a single organism

is typically small. Toxins listed on the Centers for Dis-

ease Control and Prevention’s bioterrorism threat list

are proteins of microbial or plant origin, and include

Clostridium botulinum neurotoxin, C perfringens epsilon

toxin, Staphylococcus aureus enterotoxin B, and ricin

from Ricinus communis . Additional, nonproteinaceous

toxins that may pose a threat are the trichothecene my-

cotoxins (eg, T-2 toxin) and marine toxins (eg, saxitoxin

[STX], brevetoxins, and domoic acid).

Although any of these toxins have the potential to

cause significant effects in humans or animals, their

potential as biological warfare/biological terrorism

agents varies depending on several factors. These

toxins are also clinically relevant because intoxications

occur naturally in humans and animals. The toxins in

this chapter have been selected for discussion because

of their potential for intentional use.

TRICHOTHECENE MYCOTOXINS

History

The “Yellow Rain” Controversy

Mycotoxins are metabolites of fungi produced

through secondary biochemical pathways. Various

mycotoxins are implicated as the causative agents of

adverse health effects in humans and animals that

consumed fungus-infected agricultural products. 1,2

Consequently, fungi that produce mycotoxins, as well

as the mycotoxins themselves, are potential problems

from a public health and economic perspective. The

fungi are a vast group of eukaryotic organisms, but

mycotoxin production is most commonly associated

with the terrestrial filamentous fungi referred to as

molds. 3 Various species of toxigenic fungi are capable

of producing different classes of mycotoxins, such as

the aflatoxins, rubratoxins, ochratoxin, fumonisins,

and trichothecenes. 1,2

Some of the air attacks in Laos, described as “yellow

rain,” consisted of a shower of sticky yellow liquid

that fell from the sky and sounded like rain. Other

accounts described a yellow cloud of dust or powder,

a mist, smoke, or an insect-spray–like material. More

than 80% of the attacks were delivered by air-to-sur-

face rockets and the remainder from aircraft-delivered

sprays, tanks, or bombs. 5 The use of other agents, such

as phosgene, sarin, soman, mustards, CS gas, phosgene

oxime, or BZ, has been suggested by intelligence infor-

mation and symptoms described by the victims. These

chemical agents may have been used in mixtures or

alone, with or without the trichothecenes. 5 Evidence

for, and against, the use of trichothecenes in Southeast

Asia has been fully discussed in previous texts. 6,7,8

Use in Biological Warfare

Weaponization

From 1974 to 1981 the Soviet Union and its client

states may have used trichothecene toxins 4 in Cold

War sites such as Afghanistan, Laos, and Cambodia.

These agents may have been delivered as an aerosol or

droplet cloud by aircraft spray tanks, aircraft-launched

rockets, bombs (exploding cylinders), canisters, a

Soviet handheld weapon (DH-10), and booby traps.

Alleged attacks in Laos (1975–1981) were directed

against Hmong villagers and resistance forces who

opposed the Lao People’s Liberation Army as well as

the North Vietnamese. In Afghanistan these weapons

were allegedly delivered by Soviet or Afghan pilots

against mujahideen guerrillas between 1979 and 1981.

The attacks caused at least 6,310 deaths in Laos (226

attacks); 981 deaths in Cambodia (124 attacks); and

3,042 deaths in Afghanistan (47 attacks). 5

Mycotoxins (especially T-2 toxin) have excellent

potential for weaponization because of their antiper-

sonnel properties, ease of large-scale production, and

proven delivery by various aerial dispersal systems. 5,7-11

In nanogram amounts, the trichothecene mycotoxins

(in particular T-2 toxin) cause severe skin irritation

(erythema, edema, and necrosis). 8,11-15 It is estimated

that T-2 toxin is about 400 times more potent in pro-

ducing skin injury than mustard (50 ng for T-2 vs

20 µg for mustard). 9 Lower microgram quantities of

trichothecene mycotoxins cause severe eye irritation,

corneal damage, and impaired vision. 4,5,9,16 Emesis and

diarrhea have been observed at 0.1 to 0.2 lethal doses

(LD) of trichothecene mycotoxins. 9-19

By aerosol exposure, the lethality of T-2 toxin is 10 to

50 times greater than when it is injected parenterally, 20

356

Additional Toxins of Clinical Concern

depending upon the species and exposure procedure. 21-22

With a larger dose in humans, aerosolized trichothe-

cenes may produce death within minutes to hours. 5-7

The inhaled toxicity of T-2 toxin is in the range of 200

to 5,800 mg/min/m 3 20-22 and is similar to that observed

for mustards or lewisite (range of 1,500–1,800 mg/min/

m 3 ). 23 Percutaneous lethality of T-2 toxin (median LD

[LD 50 ] in the range of 2–12 mg/kg) 9,14 is higher than

that for lewisite (LD 50 of approximately 37 mg/kg) or

mustards (LD 50 of approximately 4,500 mg/kg). 23

T-2 toxin can be produced by solid substrate fer-

mentation at approximately 9 g/kg of substrate, with

a yield of 2 to 3 g of crystalline product. 24 Several of

the trichothecene mycotoxins have been produced in

liquid culture at medium yields and large volumes

of culture for extraction. 25 A trichothecene mycotoxin

used in phase I and II cancer trials, 4,15-diacetoxyscir-

penol (DAS), was produced large scale by a procedure

considered proprietary by industry. 10 Thus, using exist-

ing state-of-the-art fermentation processes developed

for brewing and antibiotics, ton production of several

trichothecene mycotoxins would be fairly simple.

The delivery methods allegedly used in Southeast

Asia would result in a low-efficiency respiratory aero-

sol (1–5-µm particles), 26 but a highly effective droplet

aerosol could result in severe skin and eye irritation.

A National Research Council/National Academy of

Sciences expert committee estimated that the offensive

use of trichothecene mycotoxins could produce con-

centrations of approximately 1 g/m 3 in the exposure

cloud and 1 g/m 2 on the ground. 10 Much lower aerosol

concentrations could be expected to cause significant

incapacitating responses (ie, skin and eye irritation

at nano/microgram quantities) that would adversely

affect military operations.

was officially named alimentary toxic aleukia (alter-

native names in the Russian literature include septic

angina, alimentary mycotoxicosis, alimentary hemor-

rhagic aleukia, aplastic anemia, hemorrhagic aleukia,

agranulocytosis, and Taumelgetreide [staggering

grains]). 27,29 Symptoms of this disease include vomiting,

diarrhea, fever, skin inflammation, leukopenia, multiple

hemorrhage, necrotic angina, sepsis, vertigo, visual

disturbances, and exhaustion of bone marrow. 27-29,31

Extensive investigations in Russia indicated that a

toxin from Fusarium species was the causative agent

of alimentary toxic aleukia. 29,32,33 Subsequently, it was

demonstrated that T-2 toxin, a potent trichothecene

mycotoxin, was the likely agent of the disease. 33,34

Human cases of stachybotryotoxicosis (another toxic

trichothecene mycotoxin) have been reported among

farm workers in Russia, Yugoslavia, and Hungary. 35-38

This disease is caused by a mold, Stachybotrys atra, on

the hay fed to domestic animals. Symptoms of this toxi-

cosis include conjunctivitis, cough, rhinitis, burning in

the nose and nasal passages, cutaneous irritation at the

point of contact, nasal bleeding, fever, and leukopenia

in rare cases. 35,36 A macrocyclic trichothecene (satra-

toxin) is produced by Stachybotrys species, which may

be partly responsible for this toxicosis. 37-41 The only

apparent human cases of stachybotryotoxicosis in the

United States cited in the literature occurred in people

living in a water-damaged house heavily infested

with S atra. 42 Russian scientists have reported a case of

“cotton lung disease” that occurred after inhalation of

cotton dust contaminated with Dendrodochium toxicum ,

which is a fungus synonymous with Myrothecium ver-

rucaria (a natural producer of the verrucarin class of

macrocytic trichothecenes). 30,43

The “red mold disease” of wheat and barley in Japan

is prevalent in the region facing the Pacific Ocean. 16,44 In

humans, symptoms of this disease included vomiting,

diarrhea, and drowsiness. Toxic trichothecenes, includ-

ing nivalenol, deoxynivalenol, and monoacetylniva-

lenol (fusarenon-X), from F nivale were isolated from

moldy grains. 16,44 Similar symptoms were described

in an outbreak of a foodborne disease in the suburbs

of Tokyo, which was caused by the consumption of

Fusarium -contaminated rice. 10

In addition to human intoxication, ingestion of

moldy grains contaminated with trichothecenes has

also been associated with mycotoxicosis in domestic

farm animals. 30,31,44-51 Symptoms include refusal of feed,

emesis, diarrhea, skin inflammation, hemorrhage, abor-

tion, cyclic movement, stomatitis, shock, and convul-

sions. Overall, the symptoms evident in domestic farm

animals that eat food contaminated with trichothecene

mycotoxins are similar to those observed in humans.

Description of the Toxin

Natural Occurrence

Potentially hazardous concentrations of the tricho-

thecene mycotoxins can occur naturally in moldy

grains, cereals, and agricultural products. 10,16 Toxigenic

species of Fusarium occur worldwide in habitats as

diverse as deserts, tidal salt flats, and alpine mountain

regions. 10 A food-related mycotoxic disease has been

recorded in Russia from time to time, probably since

the 19th century. 27-29 In the spring of 1932, this disease

appeared in endemic form throughout several districts

of western Siberia (with a mortality rate of up to 60%).

From 1942 to 1947, more than 10% of the population in

Orenburg, near Siberia, was fatally affected by over-

wintered millet, wheat, and barley. 16,29,30 The syndrome

357

Medical Aspects of Biological Warfare

Chemical and Physical Properties

30 minutes inactivates them. 56,57 This emphasizes the

marked stability of trichothecene mycotoxins under

varying environmental conditions.

The trichothecenes make up a family of closely re-

lated chemical compounds called sesquiterpenoids. 16

All the naturally occurring toxins contain an olefinic

bond at C-9,10, and an epoxy group at C-12,13; the lat-

ter characterizes them as 12,13-epoxy trichothecenes.

The structures of approximately 150 derivatives of

trichothecenes are described in the scientific litera-

ture. 10,52,53 These mycotoxins are classified into four

groups according to their chemical characteristics. The

first two groups include the “simple” trichothecenes,

and the other two include the “macrocyclic” tricho-

thecenes. 16,30 Because of its relatively high toxicity and

availability, T-2 toxin has been the most extensively

studied trichothecene mycotoxin.

The trichothecene mycotoxins are nonvolatile,

low-molecular–weight (250–550) compounds. 53 This

group of mycotoxins is relatively insoluble in water;

the solubility of T-2 toxin is 0.8 and 1.3 mg/mL at 25°C

and 37°C, respectively. 54 In contrast, these toxins are

highly soluble in acetone, ethylacetate, chloroform,

dimethyl sulfoxide, ethanol, methanol, and propylene

glycol. 53 Purified trichothecenes generally have a low

vapor pressure, but they do vaporize when heated in

organic solvents. Extracting trichothecene mycotoxins

from fungal cultures with organic solvents results in a

yellow-brown liquid, which, if allowed to evaporate,

yields a greasy, yellow crystalline product believed to

be the yellow contaminant of yellow rain. In contrast,

highly purified trichothecenes form white crystalline

products that have characteristic melting points. 10

Trichothecene mycotoxins are stable compounds

in air and light when maintained as crystalline pow-

ders or liquid solutions. 10,54-57 When stored in sterile

phosphate-buffered saline at pH 5 to 8 and 25°C, T-2

toxin was stable for a year, with an estimated half-life

of 4 years. 54 In contrast, T-2 toxin degrades rapidly

over several days in culture medium containing fetal

bovine serum 58 or bacteria from soil or freshwater. 59

This suggests that enzymes present in serum or pro-

duced by bacteria can stimulate biotransformation

of trichothecene mycotoxins. A 3% to 5% solution of

sodium hypochlorite is an effective agent for inactivat-

ing trichothecene mycotoxins. 56,57 The efficacy of this

agent is increased by adding small amounts of alkali,

but higher concentrations of alkali or acid alone do not

destroy trichothecene activity. Thus, high pH environ-

ments are ineffective for inactivating trichothecene

mycotoxins. The US Army decontaminating agents DS-

2 and supertropical bleach inactivate T-2 toxin within

30 to 60 minutes. These mycotoxins are not inactivated

by autoclaving (at 250°F for 15 minutes at 15 lb/in 2 );

however, heating at 900°F for 10 minutes or 500°F for

Mechanism of Action

The trichothecene mycotoxins are toxic to humans,

other mammals, birds, fish, various invertebrates,

plants, and many types of eukaryotic cells in gen-

eral. 1,2,8,10,30,60-62 The acute toxicity of the trichothecene

mycotoxins varies somewhat with the particular

toxin and animal species. 8,10,43,60-63 Variations in species

susceptibility to trichothecene mycotoxins are small

compared to the divergence obtained by the diverse

routes of toxin administration. Once the trichothecene

mycotoxins enter the systemic circulation, regardless

of the route of exposure, they affect rapidly prolif-

erating tissues. 8,10,16 Oral, parenteral, cutaneous, and

respiratory exposures produce ( a ) gastric and intestinal

lesions; ( b ) hematopoietic and immunosuppressive ef-

fects described as radiomimetic in nature; ( c ) central

nervous system toxicity resulting in anorexia, lassi-

tude, and nausea; and ( d ) suppression of reproductive

organ function as well as acute vascular effects leading

to hypotension and shock. 2,10,20-22,30,60,63-68

These mycotoxins are cytotoxic to most eukaryotic

cells. 30,69,70 A number of cytotoxicity assays have been

developed that include ( a ) survival and cloning as-

says, 70,71 ( b ) inhibition of protein 69,72 and DNA 73,74 syn-

thesis by radiolabeling procedures, and ( c ) a neutral

red cell viability assay. 75 It takes a minimum of 24 to

48 hours to measure the effects of trichothecene my-

cotoxins on cell viability.

Uneo et al 76 first demonstrated that the trichothe-

cene mycotoxins inhibit protein synthesis in rabbit

reticulocytes and ascites cells. The inhibition of protein

synthesis by these mycotoxins occurs in a variety of

eukaryotic cells. 59,71,72,77,78 Similar sensitivity to T-2 toxin

was observed in established cell lines and primary cell

cultures. 59,72 Protein synthesis inhibition is observed

rapidly within 5 minutes after exposure of Vero cells

to T-2 toxin, with a maximal response noted within 60

minutes. 59 A number of studies have concluded that

the trichothecene mycotoxins interfere with peptidyl

transferase activity and inhibit either the initiation or

elongation process of translation. 77,79-81 Alterations in

trichothecene side groups can markedly affect protein

synthesis inhibition in in-vitro systems. 59,70,72,75,77

Substantial inhibition (86%) of RNA synthesis by

trichothecene mycotoxins was observed in human

(HeLa) cells, 77 but T-2 toxin had minor effects (15%

inhibition) on RNA synthesis in Vero cells. 59 In eu-

karyotic cells, blocking protein synthesis can severely

inhibit rRNA synthesis. 77 Because rRNA accounts for

358

Additional Toxins of Clinical Concern

80% of the total cellular RNA, the trichothecene-my-

cotoxin–related inhibition of RNA synthesis is prob-

ably a secondary effect linked to inhibited protein

synthesis.

Scheduled DNA synthesis is strongly inhibited in

various cell types exposed to trichothecene myco-

toxins. 59,71,82,83 In mice or rats given a trichothecene

mycotoxin, DNA synthesis in all tissues studied was

suppressed, although to a lesser degree than protein

synthesis. 83-87 Cells require newly synthesized protein

to exit G 1 and enter the S phase of the cell cycle, 88 dur-

ing which DNA is synthesized. Inhibitors of protein

synthesis prevent cells from entering S phase, thereby

blocking most DNA synthesis. 88 Thus, the pattern

of DNA synthesis inhibited by the trichothecene

mycotoxins is consistent with the primary effect of

these toxins on protein synthesis. For the most part,

trichothecene mycotoxins do not possess mutagenic

activity or the capacity to damage DNA in appropri-

ate cell models. 51

Because the trichothecene mycotoxins are amphiphi-

lic molecules, a number of investigations have focused

on various interactions with cellular membranes. 89,90

Yeast mutants with reduced plasma membrane were

more resistant than the parent strain to T-2 toxicity. 91,92

Changes in cell shape and lytic response to T-2 toxin

were observed in studies with erythrocytes, which

lack nuclei and protein synthesis. 93-96 Susceptibility to

lysis is species dependent and correlates with phos-

phatidylcholine. 95 In L-6 myoblasts, uptake of calcium,

glucose, leucine, and tyrosine was reduced within 10

minutes after exposure to a low dose of T-2 toxin. 89

These authors concluded that T-2 exerted multiple

effects on the cell membrane.

Once trichothecene mycotoxins cross the plasma

membrane barrier, they can interact with a number of

targets including ribosomes 77 and mitochondria. 92,97-101

These toxins also inhibit electron transport activ-

ity, as implied by decreased succinic dehydrogenase

activity 97,100,101 and mitochondrial protein synthesis. 98

Toxin-stimulated alteration in mitochondrial mem-

branes contributes to the effects on cellular energetics

and cytotoxicity. Although initial investigations on

the mechanism of action for trichothecene mycotoxins

suggested that protein synthesis is the principal target,

current observations indicate that the effects of these

toxins are much more diverse.

In cell-free or single-cell systems, these mycotox-

ins rapidly inhibit protein synthesis and polysomal

disaggregation. 10,51,67,102 Thus, it is postulated that the

trichothecene mycotoxins can directly react with cel-

lular components. Despite this direct effect, several

investigations have been published on the toxicokinet-

ics of the trichothecene mycotoxins. 53

Very little of the parent trichothecene mycotoxin is

excreted intact; rather, elimination by detoxification

is the result of extensive and rapid biotransformation.

The biotransformation of T-2 toxin occurs by four com-

peting pathways: (1) ester hydrolysis at the C-4, C-8,

and C-15 positions; (2) conjugation with glucuronic

acid; (3) aliphatic hydroxylation of the C-3N and C-4N

positions on the isovaleryl side chain; and (4) reduction

of the 12,13 epoxide.

Clinical Signs and Symptoms of Intoxication

The pathological effects and clinical signs can vary

with the route and type of exposure (acute single dose

vs chronic subacute doses). Local route-specific effects

include the following: ( a ) dermal exposure leads to lo-

cal cutaneous necrosis and inflammation 12,14,103-105 ; ( b )

oral exposure results in upper gastrointestinal tract

lesions 106-109 ; and ( c ) ocular exposure causes corneal

injury. 28 For the trichothecene mycotoxins, however,

many systemic toxic responses are similar regardless

of the exposure route. In contrast, the symptoms and

clinical signs of trichothecene intoxication can vary

depending on whether the exposure is acute or chronic.

For biological warfare use, an acute exposure would

be the major concern.

Dermal Exposure

Cutaneous irritations have been observed in indi-

viduals exposed to hay or hay dust contaminated with

trichothecene-producing molds. 35-38 While working

up large batches of fungal cultures from trichothe-

cene-producing organisms, workers suffered facial

inflammation followed by desquamation of the skin

and considerable local irritation. 110 Applying trichot-

hecene mycotoxins of relatively low toxicity (crotocin

and trichothcein) to the volar surface of a human fore-

arm or to the head resulted in erythema and irritation

within a few hours of exposure, followed by inflam-

mation that healed in 1 or 2 weeks. 111 The hands of

two laboratory workers were exposed to crude ethyl

acetate extracts containing T-2 toxin (approximately

200 µg/mL) when the extract accidentally got inside

their plastic gloves. 111 Even though the workers thor-

oughly washed their hands in a mild detergent within

2 minutes of contact, they experienced a burning

sensation in their fingers about 4 hours postexposure,

which increased in intensity until 8 hours after contact

with the toxin. Within 24 hours, the burning sensation

had disappeared and was replaced by numbness in

the fingers. After about 3 days, sensitivity was lost in

all exposed fingers, and by day 4 or 5, the affected skin

became hardened and started to turn white. During

359

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: