Lectures by

D. Mirza Tasawer Baig Plant Toxicology 2010

History
Mathieu Orfila is considered to be the modern father of toxicology, having given the subject its first formal
treatment in 1813 in his Traité des poisons, also called Toxicologie générale.

Theophrastus Phillipus Auroleus Bombastus von Hohenheim (1493–1541) (also referred to as Paracelsus,
from his belief that his studies were above or beyond the work of Celsus - a Roman physician from the first
century) is also considered "the father" of toxicology. He is credited with the classic toxicology maxim,
"Alle Dinge sind Gift und nichts ist ohne Gift; allein die Dosis macht, dass ein Ding kein Gift ist." which
translates as, "All things are poison and nothing is without poison; only the dose makes a thing not a
poison." This is often condensed to: "The dose makes the poison".

An even earlier writer on toxicology was Ibn Wahshiya, who wrote the Book on Poisons in the 9th or 10th
century.

Toxins and toxinology

Toxins are natural substances, or substances produced by living organisms, in contrast to toxic substances
from chemicals, which are toxicants. Living organisms producing or using toxins do so as either venoms or
poisons. Venoms are toxins, or more commonly, collections of varying toxins, that are used actively against
prey or predators, most commonly to subdue, kill and digest prey, or dissuade predators. Poisons of natural
origin, that is containing toxins and used by living organisms, are passive and generally used for defence. A
predator attempting to molest or eat a poisonous animal, plant or mushroom will suffer adverse effects from
the toxins in the poison, varying from mild discomfort to rapid death. Particularly food poisons, but also for
a few venoms, the component toxins are not produced by the deploying animal/plant, but are made by
micro-organisms and concentrated and used by the deploying animal/plant. A good example is tetrodotoxin,
used by a variety of poisonous animals and by a few venomous animals.

Toxins and toxinology is not solely focussed on adverse effects. An increasing number of toxins are
important as research tools, unlocking secrets of disease, or as diagnostic agents in hospital laboratories, or
as therapeutic agents to treat human disease, including anti-cancer agents, anti-epileptic agents, anti-clotting
agents, analgesics, anti-hypertensive drugs, to name but a few. This is a very rich field for research.

PLANT TOXIN (Phytotoxin)

Any substance produced by plants that is similar in its properties to extracellular bacterial toxin
nicotine - an alkaloid poison that occurs in tobacco; used in medicine and as an insecticide
strychnine - an alkaloid plant toxin extracted chiefly from nux vomica; formerly used as a stimulant
brucine - a bitter alkaloid poison resembling strychnine and extracted from nux vomica

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hemlock - poisonous drug derived from an Eurasian plant of the genus Conium; "Socrates refused to flee
and died by drinking hemlock"
toxin - a poisonous substance produced during the metabolism and growth of certain microorganisms and
some higher plant and animal species
mycotoxin - a toxin produced by a fungus
curare, tubocurarine - a toxic alkaloid found in certain tropical South American trees that is a powerful
relaxant for striated muscles; "curare acts by blocking cholinergic transmission at the myoneural junction"

Harmful Effects by Plant Toxins

 Skin
 Gastrointestinal System
 Cardiovascular Systems
 Nervous System
 Liver
 Reproductive Effects

Example – Jimson Weed

 Deadly nightshade plant (Atropa belladonna)

 Used in the Roman Empire and during the Middle Ages both as cure and a poison
 Women used preparations to dilate their pupils a sign of allure and beauty
 Atropine is drug responsible for effects
 Counteracts the effects of pesticides and chemical warfare agents that act by inhibiting
acetylcholinesterase

Example – Mushroom Poisoning

 Most dangerous mushrooms are the “death cap” (Amanita phalloides) or the “death angel” (Amanita
ocreata).
 Most susceptible are children less than 10 years of age
 Initial symptoms are nausea, vomiting, diarrhea and irregular heart rate
 Amatoxin, damages the liver cells causing liver and kidney failure and possibly death
 Amatoxin is very potent: only 0.1 to 0.3 mg/kg of body weight results in death

Plant Toxins – Skin

 Allergic Dermatitis – Plant Rashes, itchy skin

 Philodendron, poison ivy, cashew, bulbs of daffodils, hyacinths, tulips (antibody mediated)

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 Allergic Dermatitis – Pollen Sniffles & sneezing, runny eyes

 Ragweed (North America), Mugwort (Europe), grasses (antibody mediated)

 Contact Dermatitis Oral – Swelling and inflammation of mouth Skin – pain & stinging sensation
 Dumb cane (Dieffenbachia)Nettles (Urtica)

 Contact Dermatitis Skin – pain & stinging sensation

 Calcium oxalate crystals coated with inflammatory proteins – contain histamine,
acetylcholine

Plant Toxins – Gastrointestinal

 Direct stomach irritation - Nausea, vomiting and diarrhea

 California buckthorn (sacred bark), tung nut, horse chestnut, pokeweed

 Antimitotic (stops cell division) – Nausea, vomiting, confusion, delirium

 Lily family, glory lily, crocus, may apple
 Colchicine (gout treatment)

 Lectin toxicity – nausea, diarrhea, headache, confusion, dehydration, death

 Wisteria, castor bean (Ricinus communis)
 Ricin – block protein synthesis very toxic 5 to 6 beans can kill a child

Plant Toxins – Cardiovascular

 Digitalis like glycosides – cardiac arrhythmias

 Foxglove (Digitalis purpurea), squill, lily of the valley
 Contain glycosides that are similar to digitalis

 Heart nerves – decreased heart rate and blood pressure, general weakness
 Lily, hellebore, death camas, heath family, monkshood, rhododendron
 Alkaloids, aconitum, grayanotoxin (concentrated in honey)

 Blood vessel constriction (vasoconstriction)

 Mistletoe (berries contain toxin)
 Toxin is called phoratoxin

Plant Toxins - Nervous System I

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 Seizures
 Water hemlock, (parsley family), mint family

 Stimulation – Excitatory Amino Acids – headache, confusion, hallucinations

 Red alga (red tide), Green alga
 Mushrooms– Amanita family (fly agaric), Flat Pea (Lathyrus)

 Aberrant behavior – very excitable, muscle weakness, death

 Locoweed - Australian & Western U.S. plant

 Stimulation
 Coffee bean, tea, cola nut
 Caffeine, most widely consumed stimulant in the world

Plant Toxins - Nervous System II

 Neurotoxic – death
 Poison hemlock (Conium maculatum)
 Coniine – neurotoxic alkaloid – Poison used by Socrates

 Paralysis – demyelination of peripheral nerves

 Buckthorn, coyotillo, tullidora (U.S., Mexico)

 Atropine like effects – dry mouth, dilated pupils, confusion, hallucinations, memory lose
 Solanaceae family – jimsonweed, henbane, deadly nightshade (Atropa belladonna), angles
trumpet (atropine and scopolamine)

 Neuromuscular – mild stimulation to muscle paralysis, respiratory failure (curare), deathCoffee

bean, tea, cola nut
 Tobacco – South American – Strychnos family (curare) Blue green alga (anatonin A)

Plant Toxins – Liver

 “Hepatitis” and cirrhosis of liver - From contaminated grain

 Ragwort or groundsel
 Pyrrolizidine alkaloids – attack liver vessels – effects humans, cattle but some species
resistant

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 Liver failure and death
 Mushrooms – “Death cap” (Amanita phalloides)
 Amatoxin and phalloidin effects RNA and protein synthesis

 Liver cancer
 Fungus that grows on peanuts, walnuts, , etc…plant
 Alfaltoxins– produced by fungus in poorly stored grain

Plant Toxins – Reproductive

 Teratogen – malformations in offspring (sheep)

 Veratrum californicum – native to North America
 Veratrum – blocks cholesterol synthesis – seen offspring of mountain sheep

 Abortifacients
 Legumes (Astrogalus)
 Bitter melon seeds (Momordica)
 Swainsonine toxin – stops cell division
 Lectins - halt protein synthesis– used by humans

MYCOTOXIN

A mycotoxin (from Greek word mykes or mukos means "fungus" and Latin word toxicum means "poison")
is a toxic secondary metabolite produced by an organism of the fungus kingdom, including mushrooms,
molds, and yeasts. The term 'mycotoxin' is usually reserved for the relatively small (MW ~700), toxic
chemical products formed as secondary metabolites by fungi that readily colonize crops in the field or after
harvest. Most fungi are aerobic and are found almost everywhere in extremely small quantities due to the
minute size of their spores. They consume organic matter wherever humidity and temperature are sufficient.
One mold species may produce many different mycotoxins and/or the same mycotoxin as another species.

Where conditions are right, fungi proliferate into colonies and mycotoxin levels become high. Toxins vary
greatly in their severity. Some fungi produce severe toxins only at specific levels of moisture, temperature
or oxygen in the air. Some toxins are lethal, some cause identifiable diseases or health problems, some
weaken the immune system without producing symptoms specific to that toxin, some act as allergens or
irritants, and some have no known effect on humans. Some mycotoxins generally have more negative
impacts on farm animal populations than on humans. Some mycotoxins are harmful to other micro-
organisms such as other fungi or even bacteria; penicillin is one example.
Sourses
Mycotoxins can appear in the food chain as a result of fungal infection of crops, either by being eaten
directly by humans, or by being used as livestock feed. Mycotoxins greatly resist decomposition or being

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broken down in digestion, so they remain in the food chain in meat and dairy products. Even temperature
treatments, such as cooking and freezing, do not destroy mycotoxins.
Buildings are another source of mycotoxins.
Factors
The negative health effects of mycotoxins are a function of the concentration, the duration of exposure and
the subject's sensitivities. The concentrations experienced in a normal home, office or school are often too
low to trigger a health response in occupants.

Various wild mushrooms also contain an assortment of mycotoxins that can cause noteworthy health
problems for humans who eat them without first properly identifying the specimens as safe edibles, in such
cases sometimes causing mild to catastrophic mushroom poisoning.

BASIC PRINCIPLES OF TOXICOLOGY

Toxicology: The study of the adverse effects of a toxicant on living organisms.
Toxicology is an applied science that incorporates biology, chemistry, physiology, pathology, physics,
statistics, and sometimes immunology or ecology to help solve problems in forensic medicine, clinical
treatments, pharmacy and pharmacology, public health, industrial hygiene, veterinary science, agriculture,
and more, as well as giving basic insight into how an organism functions.

Toxicologist: A living organism who studies the nature of these adverse effects at the molecular, celllular,
organ, organ system, organism, or even community level by understanding what the agent does to the
system and what the system does to the agent.

Toxicant (Poison): Any agent capable of producing a deleterious response in a biological system.
“All substances are poisons; there is none that is not a poison. The right dose differentiates a poison
and a remedy.” Paracelsus (1493-1541)

The shape, size, and solubility of the toxicant will determine how easily it enters the body, how it will
distribute within the body, and the rate of its excretion from the body.

Risk Assessment: Quantitative estimate on the potential effects of various types of chemical exposure on
human health. RISK= HAZARD + EXPOSURE

AXIOMS OF TOXICOLOGY:

♦ There is essential uniformity in the biochemistry in similar species--among biological mechanisms in

mammals. This allows for extrapolation from animal data for predictions in humans.

♦ Any substance can provoke a dysfunction or injury at some degree of exposure—the dose makes the
poison.

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Attenuation of injury can be achieved by dilution; i.e., lowering the dose of the agent. Complications
can occur when there is exposure to more than one agent, even at non-toxic doses.

♦ There is a dosage or exposure level that has no effect on the health of animals—as measured by methods
which have a finite sensitivity to measure dysfunction or injury.

♦ Toxicological data from animal experiments can be used to assess the degree of exposure or dosage that
will not adversely affect human health. However, potential or real differences in animals or humans (as
well as variations in species) each mandate that judgmental factors be applied when extrapolating form
animal threshold doses in order to insure an adequate margin of safety for humans.

♦ The single most important factor that determines the potential toxicity of a substance is the relationship
between the concentration of the chemical at its site of action and the effect that is produced.

The Dose-Response Relationship is of paramount importance in toxicology.

Dose-Response curve: The relationship between the dose of a toxicant (dependent variable) and the
response produced (independent variable) follows a predictable pattern. As the dose of a toxicant increases,
so does the response, either in terms of the proportion of the population responding or in terms of the
severity of the graded responses. For most toxicants, at very low amounts, there will be no detectable effect
of the chemical (NOAEL: no observed adverse effect level). In the midrange of doses, the amount of
damage will increase as the dose increases (the linear 16-84% of the curve). Larger amounts of toxicant
will cause increasingly more severe biological responses until a maximum level of damage is reached.
Additional toxic effects may also appear along with increased doses, depicting both dose response and dose
effect relationships.
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0-1= no adverse effect level

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RESPONSE
2-3 = linear portion of the curve
4 = maximal response or effect
2

0 1 DOSE

The cumulative proportion of the population responding to a certain dose is plotted for each dose. A similar
S-shaped curve is produced since there can be a 10-30 fold variation within a population. If one uses
mortality as the response, the dose that is lethal to 50% of the population (LD50) can be calculated from the
generated curve. Different toxicants can be compared, and the one with the lowest LD50 is the most potent.
There are differences in between exposure routes and animals.

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Chemical LD50 Chemical LD50 (with route and
(mg/kg) animal)
Ethyl Alcohol 10,000 Caffeine 620mg/kg—oral mouse
192mg/kg—oral rat
Sodium Chloride 4,000 105mg/kg—iv rat
68mg/kg—iv mouse
Ferrous Sulfate 1,500 Chlorine (LC 50) 293ppm/1hr—rat
137ppm/1hr—mouse

EXPOSURE:

An organism must be exposed to an agent before there is a risk. The physical properties of the toxicant and
the concentration of the toxicant in the environment are important in determining the extent of the exposure.

Toxic effects in a biological system are not produced unless the agent or its metabolic breakdown
(biotransformation) products reach appropriate target sites in the body at a concentration and for a length of
time sufficient to cause toxicity. We need to define, HOW MUCH, HOW LONG, and HOW OFTEN.

Doses of Chemicals:

Dose = Amount / Animal Mass i.e. mg/kg of animal body weight

A given dose can be compared across animal species

Example: Need to administer 100mg/kg dose of the drug to a mouse, a rat, and a human.
20g mouse would get 2mg of drug
200g rat would get 20mg of drug
70kg human would get 7g of the drug

Duration of Exposure:

Acute: < 24hr usually single exposure

Subacute 1 month repeated doses
Subchronic 1-3 months repeated doses
Chronic >3months repeated doses

Toxic Effects: Acute vs Chronic

Overtime, the amount of chemical in the body can build up, it can redistribute, or it can
overwhelm repair mechanisms.
Toxicologists are most interested in what is the most common scenario, chronic exposure to low
doses.

Single Dose Repeated Dose

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Benzene CNS Depression Leukemia

Frequency of Exposure:

The frequency of the exposure affects the concentration at the target site—can build up to a steady
level--why some medications are taken three times a day vs. once a day to give the wanted effect.

Route and Site of Exposure:

Ingestion (gastrointestinal tract)

Inhalation (lungs)
Dermal / topical (skin)
Parenteral (intravenous--iv, intramuscular—im, intraperitoneal—ip)

Typical Effectiveness of Route of Exposure

iv > inhalation > ip > im > ingestion > topical

ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION OF TOXICANTS (ADME):

The toxicant may have to pass many barriers to get to its site of action

Absorption:

Absorption--the ability of a chemical agent to enter the blood.

Similar blood levels are more likely to give similar effects than similar administered doses. Blood is in
equilibrium with the other tissues and target sites.

Intravenous No limiting factors in absorption (100% bioavailable)

Inhalation Must penetrate alveolar sacs of lungs but then into capillary bed

Ingestion Requires absorption through GI tract and is subject to 1st pass effect

Intraperitoneal Like ingestion (still 1st pass effect) but does not require absorption through the GI
tract

Dermal/Topical Requires absorption through the skin

Distribution:

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Distribution—the process in which a toxicant translocates throughout the body. The blood carries the agent
to and from its site of action, storage depots, organs of biotransformation, and organs of elimination. The
rate of distribution is usually rapid, and is determined primarily by blood flow and the chemical
characteristics of the toxicant (its affinity for the tissue and the partition coefficient). The distribution of a
chemical may change over time.
Storage—DDT in Fatty tissues
Lead and Fluoride in Bone

Metabolism:
Metabolism (biotransformation)—the process by which administered toxicant (parent compounds) are
modified by the organism, usually via enzymes. The primary objective of metabolism is to make chemical
agents more water soluble and easier to excrete by

Decreasing lipid solubility  Decreased amount that reaches target

Increasing ionization  Increased rate of excretion  Decrease toxicity

In some situations, biotransformation results in the formation of reactive metabolites—Bioactivation.

Whether it is the parent compound or the metabolite, it is the active compound that does the damage.
Excretion:

Toxicants are eliminated from the body by several routes.

Urinary excretion
Water soluble products are filtered out of the blood and excreted into the urine.

Exhalation
Volatile compounds are exhaled through breathing

Biliary Excretion via Fecal Excretion

Compounds can be extracted by the liver, biotransformed, and excreted into the bile. The
bile
drains into the small intestine where the eliminated compound can be excreted into the feces.
Fecal excretion also rids the body of non-absorbed compounds which pass through the GI
tract.
MORE ON METABOLISM:

Biotransformation can occur at any point during the compound’s trek from absorption to excretion.

Biotransformation can drastically effect the rate of clearance of compounds

Without Biotransformation With Biotransformation
Ethanol 4 weeks 10mL/hr

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Phenobarbital 5 months 8hr
DDT infinity days to weeks

Key organs of Biotransformation:

LIVER (High)
Lung, Kidney, Intestine (Medium)
Others (Low)

Biotransformation Pathways
Phase I enzymes: Makes the toxicant more soluble
Phase II Enzymes: Links with a soluble agent (conjugation)
Individual Susceptibility:

Individual variation of the organism will affect the absorption, distribution, metabolism, and excretion of the
toxicant, and there fore the effect of the toxicant.

There can be a 10-30 fold difference in response to a toxicant in a population due to:

Genetics—species, strain variations, inter-individual variations

Gender
Age (young and old)
Nutritional status
Health conditions
Previous or concurrent exposure to other substances
M Y C O T O X I N
Mycotoxicoses are the animal diseases caused by mycotoxins;
Mycotoxicology is the study of mycotoxins.
All mycotoxins are of fungal origin, not all toxic compounds produced by fungi are called mycotoxins.
Mycotoxins are made by fungi and are toxic to vertebrates and other animal groups in low concentrations.
CLASSIFICATION

Mycotoxins are challenging to classify. Due to their diverse chemical structures and biosynthetic origins,
their myriad biological effects, and their production by a wide number of different fungal species,
classification schemes tend to be difficult.
o Clinicians / Pharmacologists often arrange them by the organ they affect. Thus, mycotoxins
can be classified as hepatotoxins, nephrotoxins, neurotoxins, immunotoxins, etc.
o Cell biologists put them into generic groups such as teratogens (agents causing defects in
structures during birth), mutagens, carcinogens, and allergens.
o Organic chemists have attempted to classify them by their chemical structures e.g., lactones,
coumarins etc.
o Biochemists according to their biosynthetic origins like polyketides, amino acid-derived, etc.
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o Physicians by the illnesses they cause e.g., St. Anthony's fire, stachybotryotoxicosis etc.
Mycologists by the fungi that produce them e.g., Aspergillus toxins, Penicillium toxins etc.

Classification of Mycotoxins According to the Main Organ System, They Affect

Mycotoxins can be classified on the basis of their major target organ system. These are:

Hepatotoxins

• Sporidesmin
• Aflatoxins
• Luteoskyrin
• Cyclochlorotine
• Rubratoxins
• Sterigmatocystin

Nephrotoxins

• Ochratoxin
• Citrinin

Neurotoxins

• Penitrema
• Patulin
• Citreoviridin

Cytotoxins (Alimentary Tract Toxins)

• Trichothecenes
• Diacetoxyscripenal
• Neosolaniol
• Nivalenol
• Diacetylnivalenol
• Deoxynivalenol

Estrogenic Mycotoxins

• F-2 Toxin

Other Mycotoxins

• Ergot
• Fescue
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• Lupinosis

Some mycotoxin, their sources and potential toxicities

Toxins Producing fungi Toxicities

Aflatoxin Aspergillus flavus Hepatocarcinogen
Aspergillus parasiticus and fatty liver
Citreoviridin Penicillium viridicatum Cardiac beri-beri
Citrinin Penicillium vindicatum Nephrotoxin
Cyclochlorotine Penicillium islandicum Hepatotoxin
Cytochalasin E Aspergillus clavatus Cytotoxicity
Ochratoxins Aspergillus ochraceus Hepatotoxin
Sterigmatocystin Aspergillus flavus Hepatocarcinogen
Aspergillus versicolor

Prevention and control of mycotoxins

The prevention of mycotoxins in our environment is a big task. In general, prevention of the contamination
of fungi and their mycotoxins in agricultural commodities can be divided into these following three levels.

1. Primary prevention

The step of prevention should be initially carried out before the fungal infestation and mycotoxin
contamination. This level of prevention is the most important and effective plan for reducing fungal growth
and mycotoxin production. Several practices have been recommended to keep the conditions unfavorable
for any fungal growth. These include:

• Development of fungal resistant varieties of growing plants;

• Control field infection by fungi of planting crops;
• Making schedule for suitable pre-harvest, harvest and post-harvest;
• Lowering moisture content of plant seeds, after post harvesting and during storage;
• Store commodities at low temperature whenever possible;
• Using fungicides and preservatives against fungal growth;
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• Control insect infestation in stored bulk grains with approved insecticides.

2. Secondary prevention

If the invasion of some fungi begins in commodities at early phase, this level of prevention will then be
required. The existing toxigenic-fungi should be eliminated or its growth to be stopped to prevent further
deterioration and mycotoxin contamination. Several measures are suggested as follows:

• Stop growth of infested fungi by re-drying the products;

• Removal of contaminated seeds;
• Inactivation or detoxification of mycotoxins contaminated;
• Protect stored products from any conditions which favour continuing fungal growth.

3. Tertiary prevention

Once the products are heavily infested by toxic fungi, the primary and secondary preventions would not be
then feasible. Any action would not be as effective as the practices mentioned above, since it will be quite
late to completely stop toxic fungi and reduce their toxin formation. However, some measures should be
done to prevent the transfer of fungi and their health hazardous toxins highly contaminated in products into
our daily foods and environment. For example, peanut oil extracted from poor-graded peanut seeds always
contains very high levels of aflatoxins and the oil-soluble toxin has to be eliminated by absorption and
alkalinization during oilrefining process. Only a few practices are recommended:

• Complete destruction of the contaminated products;

• Detoxification or destruction of mycotoxins to the minimal level.

Since aflatoxin is the most well-known mycotoxin ever thoroughly studied and its prevention and control
has been most successfully practiced in various countries, therefore, this paper will focus on such practices
in certain detail for the prevention and control of aflatoxins mycotoxin contamination. Successful
development will bring a great impact for the increased production of crops and safe and nutritious foods
around the world. A number of researchers have been working on A. flavus-resistant or tolerant varieties of
corn and peanut.

It has been clear that the fungal-resistance of each variety is genotypic. However, the resistance to invasion
of A. flavus has been attributed to several biochemicals, environmental and physical factors. Uncontrollable
factors could bring the failure in the utilization of selected fungal-resistant variety, as shown by laboratory
screening, in the field.

A differential pathogenic capacity of various toxigenic strains of A. flavus have been observed . Some
strains would require physical damage for their infestation and others would not. The association of
mycotoxin production and physical damage to grain and drought during grain ripening indicates that
Aspergillus spp. are weak pathogens. During long grain storage, the biochemical activity of grain is much

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reduced, while invasion of storage fungi and mycotoxin contamination would increase. More data is needed
on the biochemistry and pathogenesis of toxigenic fungi to understand and evaluate their genotype.

The germination and viability of maize seeds could be affected by attack of Aspergillus and Penicillium
species and their fungal infestation have been found to be different among maize genotypes .

Similarly, genotypes of peanut and biochemical properties of its seed such as tannin content , thin pericarp ,
small amount of cuticular wax and chemical composition of the pericarps and embryos have been shown to
inhibit fungal invasion by A. flavus and aflatoxin formation.

Recently, antifungal enzymes, chitinase and 3-glucanase, found in a number of plant seeds, may act as
defense against pathogenic fungi, since chitin and glucan are major polymeric components of many fungal
cell walls. Such polysaccharides in fungal cell wall could be enzymically hydrolysed into smaller products
resulting the damage or killing of fungal mycelia or spores. The role of these enzymes for genotype
evaluation is now being studied. It is foreseen that seeds rich in such antifungal enzymes likely resist the
infestation of fungi. If so, the seeds for breeding would be easily screened out and used a stock one.

Even there are many technical problems in searching for the "super" plant against pathogenicity, the
development of fungal-resistant plant varieties utilizing genetic resistance to mycotoxin contamination is
still possible and encouraged.

FUNGAL GROWTH INHIBITION

How to prevent growth and invasion of pathogenic fungi in agricultural commodities is very important in
preventing mycotoxin contamination. The inhibition of fungal growth can be achieved by physical,
chemical and biological treatments.

• Physical treatment. After the crops have been harvested, drying and proper storage and suitable
transportation of the commodities are of prime importance. Several flavourable factors contribute to
the growth of fungi and aflatoxin production, namely high moisture content, humid climate, warm
temperature (2540°C), insect infestation and pes damage. Many means and measures to prevention
of fungal contamination have been emphasized and practically done.
• Drying seeds and commodities to the safe moisture levels (<9% for peanut kernel, and < 13.5% for
corn).
• maintenance of the container or warehouse at low temperature and humidity.
• keep out insects and pests from the storage
• Gamma-irradiation of large-scale commodities.
• Chemical treatment with synthetic fungicides
• organic acids: acetic acid, propionic acid and butyric acid, malonic acid, benzoic acid , sorbic acid ,
lactic acid , citric acid and their sodium salts
• sodium chloride
• Benzoic acid derivatives : Onitrobenzoate, O-aminobenzoate, paminobenzoate, benzocain (ethly
aminobenzoate), ethyl benzoate, methyl benzoate and aspirin (O-acetoxy benzoic acid)

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• potassium sulfite and potassium fluoride
• dichorvos
• Fumigant: ammonia and phosphine .
• Treatment with natural products from plants or herbs.
• allicin and related substances from garlic and onion extracts
• chitosan or derivative of chitin isolated from crustacean shells
• cinnamon extract: trans-cinnamic acid, trans-cinnamaldehyde, and ferulic acid (phydroxy-3-methyl
cinnamic acid)
• clove oil
• Other herbs: thyme, star anise seeds , black and white peper . plumbago indica .

DECONTAMINATION OF MYCOTOXINS

Contaminated mycotoxins in foods and feeds should be removed, inactivated or detoxified by physical,
chemical and biological means depending on the conditions. However, the treatment has its own limitations,
since the treated products should be healthsafe from the chemicals used and their essential nutritive value
should not be deteriorated. The following methods are suggested to be applied for effective decontamination
of some mycotoxins.

Physically, fungi-contaminated seeds can be removed by hand picking or photoelectric detecting machines.
The method would consume time and Iabor or expensive.

Organic solvents (chloroform, acetone, hexane and methanol) have been used to extract aflatoxins for
agricultural products, but mainly in vegetable oil refining process.

Heating and cooking under pressure can destroy nearly 70% of aflatoxin in rice compared to under
atmospheric pressure only 50% destroyed . Dry and oil roastings can reduce about 50-70% of aflatoxin B1 .
We could show that only about 10% of total 1242 ppb of aflatoxin B. decreased in naturally contaminated
peanut by heating at upto 100°C . Since aflatoxin resists to higher temperature upto 260°C, long-time
cooking and overheating would destruct essential vitamins and amino acids in treated foods.

Ionizing radiation such as gamma-rays can stop growth of food spoilage organisms, including bacteria,
molds and yeasts. It also inactivates pathogenic organisms including parasitic worms and insect pests. It has
been reported that gamma radiation caused reduction of aflatoxin . The irradiation, however, could not
completely destroy the toxin and its mutagenicity. In our laboratory, only about 30% of total 600 ppb at
aflatoxin B1, either pure toxin or in contaminated peanut, was destroyed by 1 and 5 Mrad or gamma
irradiation . The treatment combination of gamma irradiation and ammoniation should be therefore
attempted for more aflatoxin decontamination.

Chemical treatment has been used as the most effective means for the removal of mycotoxins from
contaminated commodities. The method should be sure that the detoxification system is capable of
converting the toxin to a nontoxic derivative (s) without deleterious change in the raw product. Mutagenicity
of the treated products should be assessed. The toxicity may be checked by feeding animals including bouts,

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egg embryos, chicken, ducklings and rats. Many common chemicals have been brought to test the
effectiveness in detoxification of aflatoxin. These chemicals include the followings:

• acetic acid (C2H5OH)

• ammonia gas (NH3) or NH4OH or ammonium salts, 3-5%
• calcium hydroxide (Ca(OH)2)
• formaldehyde
• hydrogen peroxide (H2O2
• methylamine (CH3-NH2)
• ozone gas
• phosphoric acid (H3PO4)
• phosphine gas (PH3), very highly toxic!
• sodium bicarbonate (NaHCO3)
• sodium bisulfite (NaHSO3)
• sodium bisulfite (NaOH)
• sodium hypochlorite (NaOCI)

Aspergillus
Aspergillus is a large genus, with more than 200 species. Aspergillus species are of very common
occurrence in the environment, principally in soils and decaying vegetation, but a number of species are also
closely associated with human foods, particularly cereals and nuts. Many species are xerophilic, and capable
of spoiling foods only just above safe moisture limits. The most significant mycotoxigenic species are A.
flavus and A. parasiticus, which make aflatoxins, A. ochraceus, which makes ochratoxin, and A. versicolor,
which produces sterigmatocystin. These species are treated below: A. flavus and A. parasiticus are very
closely related, and are treated together.

CLASSIFICATION OF ASPERGILLUS

Kingdom: Fungi
Phylum: Deuteromycota
Class: Eurotiomycetes
Order: Eurotiales
Family: Trichocomaceae
Genus: Aspergillus
Species about 200

Mycotoxins produced by species of Aspergillus are :

 Aflatoxins
 Ochratoxin
 Citrinin
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 Patulin

Aflatoxins are a type of mycotoxin produced by Aspergillus species of fungi, such as A. flavus and A.
parasiticus. The term aflatoxin refers to four different types of mycotoxins produced, which are B1, B2, G1,
and G2. Aflatoxin B1, the most toxic, is a potent carcinogen and has been directly correlated to adverse
health effects, such as liver cancer, in many animal species. Aflatoxins are largely associated with
commodities produced in the tropics and subtropics, such as cotton, peanuts, spices, pistachios and maize.

Ochratoxin is a mycotoxin that comes in three secondary metabolite forms, A, B, and C. All are produced
by Aspergillus and Penicillium species. The three forms differ in that Ochratoxin B (OTB) is a
nonchlorinated form of Ochratoxin A (OTA) and that Ochratoxin C (OTC) is an ethyl ester form Ochatoxin
A. Aspergillus ochraceus is found as a contaminant of a wide range of commodities including beverages
such as beer and wine. Aspergillus carbonarius is the main species found on vine fruit, which releases its
toxin during the juice making process. OTA has been labeled as a carcinogen and a nephrotoxin, and has
been linked to tumors in the human urinary track, although research in humans is limited by confounding
factors.

Citrinin is a toxin that was first isolated from Penicillium citrinum, but has been identified in over a dozen
species of Penicillium and several species of Aspergillus. Some of thesee species are used to produce human
foodstuffs such as cheese (Penicillium camemberti), sake, miso, and soy sauce (Aspergillus oryzae). Citrinin
is associated with yellow rice disease in Japan and acts as a nephrotoxin in all animal species tested.
Although it is associated with many human foods (wheat, rice, corn, barley, oats, rye, and food colored with
Monascus pigment) its full significance for human health is unknown. Citrinin can also act synergistically
with Ochratoxin A to depress RNA synthesis in murine kidneys.

Patulin is a toxin produced by the P. expansum, Aspergillus, Penicillium, and Paecilomyces fungal species.
P. expansum is especially associated with a range of moldy fruits and vegetables, in particular rotting apples
and figs. It is destroyed by the fermentation process and so is not found in apple beverages, such as cider.
Although patulin has not been shown to be carcinogenic, it has been reported to damage the immune system
in animals.

Claviceps purpurea

Claviceps purpurea is a fungus that grows on the ears of rye and related cereal and forage plants.
Consumption of grains or seeds contaminated with the fruiting structure of this fungus, the ergot sclerotium,
can cause ergotism in humans and other mammals. C. purpurea most commonly affects outcrossing species
such as rye (its most common host), as well as triticale, wheat and barley. It affects oats only rarely.

Ergot refers to a group of fungi of the genus Claviceps (ergot fungi). The most prominent member of this
group is Claviceps purpurea. This fungus grows on rye and related plants, and can cause ergotism in
humans and other mammals consuming seeds contaminated with the fruiting structure of this fungus, called
an ergot sclerotium. There are about 50 known species of Claviceps, most of them in the tropical regions.
Economically important species are C. purpurea (parasitic on grasses and cereals), C. fusiformis (on pearl
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millet, buffel grass), C. paspali (on dallis grass), and C. africana (on sorghum). C. purpurea most
commonly affects outcrossing species such as rye (its most common host), as well as triticale, wheat and
barley. It affects oats only rarely.

Kingdom: Fungi
Division: Ascomycota
Class: Sordariomycetes
Subclass: Hypocreomycetidae
Order: Hypocreales
Family: Clavicipitaceae
Genus: Claviceps
Species purpurea

Claviceps purpurea and Ergot poisoning

 Highly Stable molecule

 Also Heat Stable
 withstands long storage
 withstands brewing processes
 withstands boiling
Target organs
 Brain
 Nerve
 muscle tissue
 circulatory
 Endorcrine systems

Symptoms
 Flu like symptoms
 mental confusion and hallucinations
 limb paralysis
 abortion
 seizures
 dry gangrene due to vasoconstriction
 death

Controlling C. purpurea
1. Most wheat seed planted should be genetically engineered to be resistant to this mold
2. Use substances to prevent the growth of molds like :
 Ammonia

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 Propionic acid
 Chlorine vapours
 Sorbic acid

BACTERIAL TOXIN
A bacterial toxin is produced by bacteria.

 Exotoxins are generated by the bacteria and actively secreted.

 Endotoxins are part of the bacteria itself. Usually, endotoxin is part of the bacterial outer membrane,
and it is not released until the bacteria is killed by the immune system.

Clostridium botulinum toxin

Bacterial toxins produced by Clostridium botulinum are said to be

botulinal toxin , botulinum toxin or botulinus toxin. It is an exotoxin that produces paralysis by blocking
the release of acetylcholine in the central nervous system.
There are seven immunologically distinct types (A–G).
Type A is used therapeutically to inhibit muscular spasm in the treatment of dystonic disorders such as
blepharospasm and strabismus, as well as to treat wrinkles of the upper face.
Type B is used to treat cervical dystonia.
Clostridial toxin produced by species of Clostridium, including those causing botulinus, gas gangrene, and
tetanus.

Botulism
Botulism is a rare, but very serious condition caused by toxin produced by bacteria called Clostridium
botulinum. The toxins that this bacterium produces are among the most poisonous substances known to
humans.

Three forms of naturally occurring botulism affecting humans. These are

• Foodborne
• Woun
• Intestinal.
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All forms of botulism result from absorption of botulinum toxin into the bloodstream. The toxin does
not penetrate intact skin. Inhalational botulism is a man-made form of disease that results from inhaling
aerosolized botulinum toxin.

Staphylococcus aureous toxin

S. aureus is a facultative anaerobic Gram-positive coccus. Various types of skin eruptions and
inflammations (boils acne styes etc) and wounds can harbour large numbers of these micro-organisms.
There are 11 different toxins have been identified and implicated in foodborne illness.

Toxins
Depending on the strain, S. aureus is capable of secreting several toxins, which can
be categorized into three groups. Many of these toxins are associated with specific
diseases.
(A) Pyrogenic toxin superantigens (PTSAgs)
They have superantigen activities that induce toxic shock syndrome (TSS). This
group includes the toxin TSST-1, which causes TSS associated with tampon use.
The staphylococcal enterotoxins, which cause a form of food poisoning, are
included in this group.
(B) Exfoliative toxins (EF)
These toxins are implicated in the disease staphylococcal scalded-skin
syndrome (SSSS), which occurs most commonly in infants and young children. It
also may occur as epidemics in hospital nurseries. The protease activity of the
exfoliative toxins causes peeling of the skin observed with SSSS.
(C) Other toxins
Staphylococcal toxins that act on cell membranes include alpha-toxin, beta-
toxin, delta-toxin, and several bicomponent toxins. The bicomponent
toxin Panton-Valentine leukocidin (PVL) is associated with severe
necrotizing pneumonia in children. The genes encoding the components of PVL
are encoded on a bacteriophage found in community-associated MRSA strains.

Microcystis aerugnosa
Globally the most frequently found cyanobacterial toxins in blooms from fresh and brackish waters are the cyclic
peptide toxins of the microcystin and nodularin family. They pose a major challenge for the production of safe
drinking water from surface waters containing cyanobacteria with these toxins.
About 60 structural variants of microcystins have been characterised so far from bloom samples and isolated
strains of cyanobacteria.

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Microcystins have been characterised from planktonic Anabaena, Microcystis, Oscillatoria (Planktothrix),
Nostoc, and Anabaenopsis species, and from terrestrial Hapalosiphon genera. Nodularin has been characterised
only from Nodularia spumigena.
Some are :
Nodularins
Nodularins
Anatoxin-a
Cylindrospermopsin
Saxitoxins

Polyketides are secondary metabolites from bacteria, fungi, plants, and animals. Polyketides are
biosynthesized by the polymerization of acetyl and propionyl subunits in a similar process to fatty acid
synthesis (a Claisen condensation). They are the building blocks for a broad range of natural products or are
further derivatized.

Polyketides are structurally a very diverse family of natural products with diverse biological activities and
pharmacological properties. Polyketide antibiotics, antifungals, cytostatics, anticholesterolemics,
antiparasitics, coccidiostatics, animal growth promoters and natural insecticides are in commercial use.

Teratology generally refers to disfiguring birth defects or malformations.

Ergotism is the effect of long-term ergot poisoning, traditionally due to the ingestion of the alkaloids
produced by the Claviceps purpurea fungus which infects rye and other cereals, and more recently by the
action of a number of ergoline-based drugs. It is also known as ergotoxicosis, ergot poisoning and Saint
Anthony's Fire.

Stachybotryotoxicosis

A type of mycotoxicosis seen in horses and cattle following ingestion of hay and fodder overgrown by the
fungus Stachybotrys atra; may also occur in persons exposed to hay either by inhalation or by absorbing the
toxin through the skin, and is manifested by skin rash, pharyngitis, and mild leukopenia.

“OR”

A serious and sometimes fatal intoxication chiefly affecting domestic animals (as horses) that is due to
ingestion of a toxic substance produced by a mold (Stachybotrys chartarum)

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