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					     Mycotoxins
    Introduction


1
    Mycotoxins are secondary metabolites (secondary metabolite:                                          A
    compound that is not necessary for growth or maintenance of cellular functions but is synthesized,
    generally, for the protection of a cell or micro-organism, during the stationary phase of the growth
                                                  .) of fungi
    cycle. Many are used in foods, pharmaceuticals, and other industrial applications
    that are recognized as toxic to other life forms.

1.Fungal growth
• a. Field fungi : fungi that attack plants that grow in the field (occurring prior to harvest)
    grow under special conditions. (Fusarium)
• b. Storage fungi : Storage fungi usually invade grain or seed during storage and are
    generally not present in large quantities before harvest in the field. The most common storage fungi
    are species of Aspergillus and Penicillium. Contamination occurs through spores
    contaminating the grain as it is going into storage from the harvest. The development of fungi is
    influenced by the:
     –   Moisture content of the stored grain
     –   Temperature
     –   Condition of the grain going into storage
     –   Length of time the is grain stored and
     –   Amount of insect and mite activity in the grain




2
2. Characteristics of mycotoxin induced
disease

    a. not transmitted among animals
    b. Pharmaceutical treatment does not alter the
      course of disease
    c. Mycotoxicosis most often presents as a
      uncertain, sub-acute or chronic condition




3
    3.Treatment of mycotoxin-
         induced disease

a. For most mycotoxins, there is no
specific treatment or antidote
b. Supplement with vitamins & selenium
may be helpful, and provision of adequate
high-quality protein


4
    4.Prevention of mycotoxin-
         induced disease
a. Avoiding
b. Diluting
c. Cleaning
d. Testing
e. Drying
f. Adding (organic acids will prevent
    mold growth)

5
                    A- Aflatoxin


• 1. Sources :
Aspergillus flavus & A.paraciticus :

      Corn, peanuts


• 2. Factor favoring production of aflatoxins

      a. Temperature : 25-30 ๐c
      b. Grain moisture
  6
    3. Chemical characteristics

• Exhibit intense blue or green
  fluorescence under UV.
     : aflatoxins B1, B2, G1 and G2
also
     : aflatoxin M1 is a metabolites of
  AFB1 found in animal urine, milk or
  tissues.

7
    5.Mechanism of toxicologic
            damage
              Also called steatosis, fatty liver can be a temporary or long-term condition, which is
              not harmful itself, but may indicate some other type of problem. Left untreated, it can
              contribute to other illnesses. It is usually reversible once the cause of the problem is
              diagnosed and corrected. The liver is the organ responsible for changing fats eaten in
              the diet to types of fat that can be stored and used by the body. Triglycerides are one
              of the forms of fat stored by the body and used for energy and new cell formation. The
              break down of fats in the liver can be disrupted by alcoholism, malnutrition, pregnancy,
              or poisoning. In fatty liver, large droplets of fat, containing mostly triglycerides, collect
              within cells of the liver. The condition is generally not painful and may go unnoticed for
              a long period of time. In severe cases, the liver can increase to over three times its normal size and
              may be painful and tender




8
• a. Loss of enzyme
• b. Lack of formation of lipid acceptor
  protein in liver
• c. Decreased cellulose digestion,
  volatile fatty acid formation &
  proteolysis (breakdown of proteins )



• d. Necrosis



9
                 6.Toxicity

• a. Young animals are more susceptible than
  adult.


• b. Nutrition deficiency increase
  susceptibility


  10
              7. Diagnosis


• Clinical sign : decreased growth rate,
  reduced feed efficiency,,, mild anemia,
  and increased susceptibility to infectious
  disease.




 11
       8.Treatment & Prevention


• a. Detoxification : Hydrated sodium
  calcium aluminosilicate (HSCAS) can
  absorb aflatoxins
• b. Supportive : Vitamin .E & selenium
• c. Prevention
      - Mold inhibitor
   - Treatment of grain with anhydrous
 ammonia for 10-14 days.
 12
               B- Zearalenone

• 1. Sources : Fusarium roseum
( F.graminearum ):
         corn, wheat, barley, oats


• 2. Factor favoring production
      a. High moisture 22% - 25%
      b. Alternating high and low temp. (7-21 ๐c)
 13
     3. Mechanism of toxicological damage
     a. initiating specific RNA synthesis
     b. Function as a weak estrogen.



                   4.Toxicity

     a. Swine are most susceptible
     b. low for all effects except reproductive
     function.

14
                C-Ergot

1.Source :
Claviceps purpurea :
         barley, wheat & oats

2. Factor favoring :
          Warm & humid

 15
           3.Mechanism of toxic
• a. potent initiators of contraction in smooth muscle
• b. mimic the action of dopamine.

4.Clinical sign
a. necrosis of the feet, ears and tail
b. increased temperature., pulse & respiration rate
c. lactation does not occur
d. hyper-excitability & tremors
e. heat intolerance in cattle

 16
               E.Treatment

     a. animals should be provided with a
     warm, clean, stress-free environment

     b. Control secondary bacterial
     infection

     c. milk supplement

17
       D- Ochratoxin & Citrinin
• 1.Sources : Aspergillus orchraceus &
• Penicillium viridicatum
• 2. Mechanism of toxic :
            target the renal proximal tubule
• - Disrupt protein synthesis
• -Bind strongly to protein (albumin)
• -Interfere with synthesis of tRNA & mRNA
• -Disrupt carbohydrate metabolism
• -Increase the generation of free radical
 18
                4.Clinical sign


• a. Acute : vomiting, diarrhea, dehydration &
  depression

• b. Subacute to chronic : weight loss, feed efficiency,
  & dehydration. Immunosupression, teratogenicity,
  carcinogenesis & hemorrhage


  19
     Mycotoxins


20
                       Mycotoxin
Mycotoxin is a convenient generic term describing the toxic
secondary metabolites produced by fungi. “Myco” means
fungal (mold) and “toxin” represents poison.

They encompass a considerable variety of low molecular
weight compounds with diverse chemical structures and
biological activities.
Some mycotoxins could also be toxic to plants or other
microorganisms; but these compounds are not classified as
antibiotics of fungal origin.

Like most microbial secondary metabolites, the benefit of
mycotoxins for the fungi themselves is still not clearly defined.
21
• In considering the effects of mycotoxins on
  animals, it is important to distinguish
  between “mycotoxicosis” and “mycosis.”:

• Mycotoxicosis is used to describe the
  action of mycotoxin(s) and is frequently
  mediated through a number of organs,
  notably the liver, kidney, lungs, and the
  nervous, endocrine, and immune systems.

• Mycosis” refers to a generalized invasion
  of living tissue(s) by growing fungi.
22
• Due to their diverse chemical structures,
  mycotoxins may exhibit a number of
  biological effects, including both acute and
  chronic toxic effects as well as carcinogenic,
  mutagenic, genotoxic, and immunotoxic
  effects.

• The interaction of mycotoxins with cellular
  macromolecules plays a dominant role in
  their toxic actions. Recent studies on the
  effect of mycotoxins on apoptosis have
  further revealed their mode of action at the
  cellular level.
 23
Historical
• Modern mycotoxicology was not developed until the discovery
  of aflatoxins in the early 1960s as the causative agent in the
• peanut meal causing the “Turkey X” disease that killed more
  than 10,000 turkeys fed with the contaminated meal.

• Because aflatoxins are a series of highly potent carcinogens
  produced by commonly occurring Aspergillus flavus and A.
  parasiticus, research has focused new attention on mycotoxins.


• In the last 40 years, many new mycotoxins have been identified
  and characterized, and their biosynthetic origin in various fungi
  elucidated. It has been estimated that at least 25% of the
  world’s agricultural product is contaminated with mycotoxins
  and certain diseases have been linked to ingestion of food and
  feed contaminated with mycotoxins.
   24
Economic Impact of Mycotoxin Contamination

• The most obvious negative economic impact of
  mycotoxins is an outright loss of crops and affected
  animals.
• Also, humans may encounter severe health hazard or
  high mortality rates in countries with less regulation or
  monitoring programs.

• Thus, the negative economic impact resulting from
  mycotoxin contamination is certainly very significant
  and estimated to be $932 million annually.

   25
PRODUCTION OF MYCOTOXINS BY TOXICOGENIC
FUNGI
Invasion by fungi and production of mycotoxins in commodities can occur
under favorable conditions in the field, at harvest, and during processing,
transportation and storage
Fungi that are frequently found in the field include: A. flavus, Alternaria
longipes, A. alternata, Claviceps purpura, Fusarium verticillioides
(previously called moniliforme), F. graminearum, and a number of other
Fusarium spp.
Species most likely introduced at harvest include:
 F. sporotrichioides, Stachybotrys atra, Cladosporium sp., Myrothecium
verrucaria, Trichothecium roseum, as well as A. alternata.

Most penicillia are storage fungi. These include: Penicillium citrinum, P.
cyclopium, P. citreoviride, P. islandicum, P. rubrum, P. viridicatum, P. urticae,
P. verruculosum, P. palitans, P. puberulum, P. expansum, and P. roqueforti.

All of which are capable of producing mycotoxins in grains and foods.
  26
• Other toxicogenic storage fungi are: Aspergillus.
  parasiticus, A. flavus, A. versicolor, A. ochraceus, A.
  clavatus, A. fumigatus, A. rubrum, A. chevallieri,
  Fusarium verticillioides, F. tricinctum, F. nivale, and
  several other Fusarium spp.


• It is apparent, most of the mycotoxin producing fungi
  belong to three genera: Aspergillus, Fusarium, and
  Penicillium. However, not all species in these
  genera are toxicogenic

  27
Factors Affecting Mycotoxin Production

• Genetics and environmental and nutritional factors
  greatly affect the formation of mycotoxins.

• Depending on the susceptibility of the crop, geographic
  and seasonal factors, as well as cultivation, harvesting,
  storage, and transportation practices, mycotoxins are
  found worldwide.
• In the field, weather conditions, plant stress,
  invertebrate vectors, species and spore load of
  infective fungi, variations within plant and fungal
  species, and microbial competition all significantly
  affect mycotoxin production.
  28
                                     Continue Factors Affecting…….


• Physical factors such as time of exposure,
  temperature during exposure, humidity, and
  extent of insect or other damage to the
  commodity prior to exposure determine
  mycotoxin contamination in the field or during
  storage.
• Chemical factors including the nutritional
  status of the crops or chemicals (such as
  fungicides) used in crop management could
  affect fungal populations, and consequently
  toxin production
 29
                                                                                                      Continue Factors Affecting…….




• In general, mycotoxins are optimally produced
  at 24–28C, but some toxins such as T-2 toxin
  is maximally produced at 15C.

• Contamination during crop storage may be
  affected by changes in temperature and water
  activity, that allow ecological succession of
  different fungi as water activity and temperature
  of stored grain changes.

          Water activity = It is defined as the vapor pressure of water above a sample divided by that of pure water at the
  30      same temperature;
                                     Continue Factors Affecting…….




• During storage and transportation, water
  activity (aw), temperature, crop damage, and a
  number of physical and chemical factors, such
  as aeration (O2, CO2 levels), types of grains,
  pH, and presence or absence of specific
  nutrients and inhibitors are important.




 31
   Aflatoxins
                                          A           B
                                                  R
Chemical structure of flatoxins                   3
                                              R           R
                                              1       R   1
                                                      4
                                                  R
(A) The B-type aflatoxins are             C       2   D
                                              R           R
    characterized by a cyclopentane E-                    1
                                              1
    ring. These compounds have a
    blue fluorescence under long-
    wavelength ultraviolet light.

(B) The G-type aflatoxins, with a green
   fluorescence, have a xanthone ring
   in place of the cyclopentane.

(C) Aflatoxins of the B2 and G2 type
   have a saturated bis-furanyl ring.
   Only the bis-furan is shown.

(D) Aflatoxin of the B1a and G1a type
    have a hydrated bis-furanyl
    structure.
   32
• At least 16 structurally related toxins in this group
  are produced by Asparagillus flavus and A.
  parasiticus and infrequently by A. pseudotamarii and
  A. nominus
• A. ochraceoroseus has also been found to
  produce aflatoxins

• The optimal temperatures and water activity (aw) for
  the growth of A. flavus and A. parasiticus are around
  35–37C (range from 6–54C) and 0.95 (range from
  0.78–1.0), respectively; whereas for aflatoxin
  production, they are 28–33C and 0.90–0.95 (range
  from 0.83–0.97), respectively.
 33
• Aflatoxin B1 is most toxic in this group and is
  one of the most potent naturally occurring
  carcinogens).



• Other significant members of the aflatoxin
  family, such as M1 and M2, are metabolites
  of AFB1 and AFB2, respectively, and
  originally isolated from bovine milk.

 34
    Natural Occurrence
• Aflatoxins have been found in corn, peanuts ‫ فول سوداني‬and
  peanut products, cotton seeds, peppers, rice, pistachios, ‫فستق‬
  tree nuts, pumpkin ‫ قرع‬seeds, sunflower seeds and other oil
  seeds, copra, ‫ جوز هند‬spices, and dried fruits (figs, raisins).
• Among these products, frequent contamination with high
  levels of AF in peanuts, corn, and cottonseed, mostly due to
  infestation with fungi in the field, are of most concern.
• Soybeans ‫ ,الصويا‬beans ‫ ,الفاصولياء‬pulses (Pea)‫ ,البازالء‬cassava
  ‫ ,منيهوت‬sorghum ‫ , الذرة‬millet ‫ ,الدخن‬wheat ‫ ,القمح‬oats ‫ ,القطن‬barley
  ‫ ,الشعير‬and rice‫ رز‬are resistant or only moderately susceptible
  to AF contamination in the field.
•    It should be reiterated that resistance to AF contamination in
    the field does not guarantee that the commodities are free of
    AF contamination during storage. Inadequate storage
    conditions, such as high moisture and warm temperatures
    (25–308C), can create conditions favorable for the growth of
    fungus and production of AF.
    35
         Toxic Effects
• Aflatoxins are mutagenic, teratogenic, and hepatocarcinogenic.

•  Aflatoxin B1 is one of the most potent naturally occurring
  carcinogen, extensive research was primarily done on this
  toxin. The main target organ of AF is the liver.
• AFB1 also affects other organs and tissues including the lungs
  and the entire respiratory system.
•   For the carcinogenic effects, rats, rainbow trout, monkeys, and
    ducks are most susceptible and mice are relatively resistant.

• Consumption of AFB1-contaminated feed by dairy cows results
  in the excretion of AFM1 in milk. AFM1, a hydroxylated
  metabolite of AFB1, is about 10 times less toxic than AFB1; but
  its presence in milk is of concern for human health.

    36
Impact on Human Health
• Whereas AFB1 has been found to be a potent carcinogen in
  many animal species, the role of AF in carcinogenesis in
  humans is complicated by hepatitis B virus (HBV) infections in
  humans).



• Epidemiological studies have shown a strong positive
  correlation between AF levels in the diet and primary
  hepatocellular carcinoma.


•     Since multiple factors are important in carcinogenesis and
     environmental contaminants such as AFs and other
     mycotoxins may, either in combination with HBV or
     independently.
37
 Ochratoxins
• Ochratoxins, are produced by a number of fungi in
  the genera Aspergillus and Penicillium. The largest
  amounts ochratoxins are made by A. ochraceus and
  P. cyclopium.
• A. ochraceus and P. viridicatum (reclassified as P.
  verrucosum), two species that were first reported as
  ochratoxin A (OA) producers, occur most frequently
  in nature.
• Other fungi, such as Petromyces alliceus, A.
  citricus, and A. fonsecaeus (both in A. niger group),
  have also been found to produce OA. Most of the
  OA producers are storage fungi and preharvest
  fungal infection.
 38
• Although most OA producers can grow in a range from 48C to
  37C and at aw as low as 0.78, optimal conditions for toxin
  production are narrower with temperature at 24–25C and aw
  values .0.97.

• Ochratoxins are produced primarily in cereal grains (barley,
  oats, corn, wheat) and mixed feed during storage in temperate
  climatic conditions, with levels higher than 1 ppm being
  reported.
• OA has been found in other commodities, including beans,
  coffee, nuts, olives, raisin, cheese, fish, pork, milk powder, fruit
  juices wine beer, peppers.

•    OA can be carried through the food chain because of the
    presence of OA residues in animal products as result of its
    binding with serum albumin. Natural occurrence of OA in
    kidneys, blood serum, blood sausage.
    39
Structure of the ochratoxins. These metabolites form different classes depending on the nature of the

amide group (a–c), and the presence or absence of a chlorine moiety at R2 in the phenyl group   .
40
     Ochratoxin A, the most toxic member of this
     group of mycotoxins, has been found to be a
     potent nephrotoxin causing kidney damage
     as well as liver necrosis and enteritis in
     many animal species
 The OA inhibits carboxypeptidase A, renal
 phosphoenolpyruvate carboxykinase,
 phenylalaninetRNA synthetase, and phenylalanine
 hydroxylase activity.

Formation of free radicals has been considered as
one of the mechanisms for the carcinogenic/toxic
effects of OA.
41
Fumonisins
Fumonisins (Fm) are a group of toxic metabolites produced primarily by F.
verticillioides, F. proliferatum and other related species readily colonize corn
all over the world. Although F. anthophilum, F. nupiforme, and F. nygamai
are capable of producing Fms.
More than 11 structurally related Fms (B1, B2, B3, B4, C1, C4, A1, A2, etc.),
have been found since the discovery of FmB1.
Fumonisins are most frequently found in corn, corn-based foods, and other
grains (such as sorghum and rice). The level of contamination varies
considerably with different regions and year, ranging from negligible to more
than 100 ppm; but is generally reported to be between 1 and 2 ppm.

FmB1 is the most common Fm in naturally contaminated samples; FmB2
generally accounts for 1/3 or less of the total. Although production of the
toxin generally occurs in the field, continued production of toxin during
postharvest storage also contributes to the overall levels.

 42
Toxicologic Effects
Fumonisin B1 is primarily a hepatotoxin and carcinogen in rats. Feeding
culture material from F. verticillioides or pure FmB1 to rats resulted in
‫تليف كبدي‬
cirrhosis and hepatic nodules, carcinoma. Kidney is also a target organ.
Mechanistically, Fms are inhibitors of ceramide synthase
(sphinganine/sphingosine N-acyltransferase), a key enzyme involved in the
biosynthesis of sphingolipids, which are heavily involved in cellular
regulation, including cell differentiation, mitogenesis and apoptosis




The ability of FmB1 to alter gene expression and signal transduction
pathways are considered necessary for its carcinogenic and toxic effects.
FmB1 is a good example of an apparently non-genotoxic (non-DNA
reactive) agent producing tumors through the regulation of apoptosis
  43
Trichothecenes (TCTCs)
Several species of Fusaria infect corn, wheat, barley, and rice.
Under favorable conditions, they elaborate a number of different types of
mycotoxins (look figure).
(TCTCs) are generally classified as macrocyclic (Type C) or nonmacrocyclic
(Types A and B). Although more than 100 TCTCs have been identified, only a
few frequently found in foods and feeds are potentially hazardous to human
and animal health.


                           Trichothecenes




Other fungal genera elaborate TCTCs are: Myrothecium, Trichoderma,
Trichothecium, Cephalosporium, Verticimonosporium, and Stachybotrys. In
addition to fungi, extracts from a Brazilian shrub, Baccharis megapotamica,
also contain macrocyclic TCTCs. The term TCTCs is derived from
trichothecin, the first compound isolated in this group.
  44
The TCTC mycotoxicoses affect many organs, including the gastrointestinal
tract, hematopoietic, nervous, immune, hepatobiliary, and cardiovascular
systems.

Mechanistically, inhibition of protein synthesis is one of the earlier events in
manifestation of TCTC toxic effects and they act at different steps in the
translation process. Inhibitory effects of these mycotoxins vary considerably
with the chemical structure of the side chain.


 a) T-2 toxin,
T-2 toxin, a highly toxic type A TCTC, is produced by F.
tricinctum, F. sporotrichioides (major), F. poae,
F. sulphureum, F. acuminatum, and F. sambucinum.
Unlike most mycotoxins, which are usually synthesized
near 25C, the optimal temperature for T-2 toxin
production is around 15C.
   45
Almost all the major TCTCs, including T-2 toxin,
are cytotoxic and cause hemorrhage, edema,
and necrosis of skin tissues.

 b) Deoxynivalenol (DON)
The DON is a major type B TCTC mycotoxin produced
by F. graminearum (major) and other related fungi
such as F. culmorum and F. crookwellense. Because
DON causes feed refusal and emesis in swine, the
name “vomitoxin” is also used for this mycotoxin.



  46
• Worldwide frequent natural occurrence of DON in
  cereal grains has been reported. Contamination of this
  toxin in corn and wheat is generally high.
• Also, contamination of barley, oats, sorghum, rye,
  safflower seeds, and mixed feeds has also been
  reported.
• Although inadequate storage may lead to the
  production of some TCTC mycotoxins, infestation of
  fusaria in wheat and corn in the field is of most
  concern for the DON problem.

• With wet and cold weather during maturation,grains
  are especially susceptible to F. graminearum infection,
  which causes so-called “scabby wheat” and
  simultaneously produces the toxin. The optimal
  temperature for DON production is about 248C.
  47
• Toxicologically, DON induces anorexia and emesis both in
  humans and animals. Swine are most sensitive to feed
  contaminated with DON. Whereas most TCTCs are
  immunosuppressors, DON is a hyperinducer of cytokines.




 48
• Other Selected Mycotoxins
• In addition to the mycotoxins discussed above, a number of other
  mycotoxins occur naturally.

• Other Mycotoxins Produced by Aspergillus:

• Sterigmatocystin (ST) is a naturally occurring hepatotoxic and
  carcinogenic mycotoxin produced by fungi in the genera Aspergillus,
  Bipolaris, and Chaetomium as well as P. luteum.

• Structurally related to AFB1 ST is known to be a precursor of AFB1.

• ST is a mutagen and genotoxin and has been found in cereal grains
  (barley, rice, and corn), coffee beans, and cheese.




      Structure of sterigmatocystin. The bis-furanyl structure is similar to
      that of the aflatoxins except that the E-ring is a substituted phenol.

 49
• A. terreus and several other fungi (e.g., A. flavus and A.
  fumigatus) have been found to produce the tremorgenic
  toxins, territrems, aflatrem, and fumitremorgin.


•     A. terreus, A. fumigatus, and Trichoderma viride also
     produce gliotoxin, In addition, A. flavus, A. wentii, and
     A. oryzae, are capable of producing nitropropionic acid
     (NPA), a mycotoxin causing apnea, convulsions,
     congestion in lungs and liver.

• Production of NPA in sugarcanes by Arthrinium sacchari,
  Arth. saccharicola, and Arth. Phaeospermum has been
  found to be involved in fatal food poisoning in humans



50
Other Mycotoxins Produced by Penicillium
Penicillia produce many mycotoxins with diverse toxic effects.
Cyclochlorotine, luteoskyrin (LS), and rugulosin (RS) have long been
considered to be possibly involved in the yellow rice disease during the
Second World War. They are hepatotoxins. Several other mycotoxins,
including patulin (PT) penicillic acid (PA) citrinin (CT), cyclopiazonic acid
(CPA, citreoviridin, and xanthomegnin, which are produced primarily by
several species of Penicillia .PT and PA are produced by many species in
the genera Aspergillus and Penicillium. Byssochlamys nivea also produces
PT




                                              penicillic acid
        Patulin
  51
                           Chemical structure of cyclopiazonic acid




 Stucture of zearalenone                    Structure of alternariol

52
• Other Mycotoxins Produced by Fusarium
• Some fusaria are capable of producing mycotoxins other than
  TCTCs and Fm. Zearalenone (ZE) a mycotoxin produced by
  the scabby wheat fungus, F. graminearum (roseum), is of most
  concern. Also called F-2, ZE is a phytoestrogen causing
  hyperestrogenic effects and reproductive problems such as
  premature onset of puberty in female animals,especially swine.
• ZE has been shown to bind with the estrogen and steroid
  receptors, and stimulates protein synthesis by mimicking
  hormonal action.
•   Zearalenone can be toxic to plants; it can inhibit seed
    germination and embryo growth at low concentrations.
• Natural contamination with ZE primarily occurs in cereal grains
  such as corn and wheat
    53
• Fusarium verticillioides and related species, also produce
  several other mycotoxins, including fusarins A-F,
  moniliformin, fusarioic, and fusaric acid, fusaproliferin and
  beauvericin.

• Although the impact of these mycotoxins on human health is
  still not known, fusarin C (FC) has been identified as a potent
  mutagen and is also produced by F. subglutinans, F.
  graminearium and several other Fusaria.

• Moniliformin, which causes cardiomyopathy in test animals,
  may be involved in the Keshan disease in humans in regions
  where dietary selenium deficiency is also a problem.

• Among many fungi, F. verticilioides is also most capable of
   reducing nitrates to form potent carcinogenic nitrosamines.
   These observations further suggest that the contamination of
   foods with this fungus could be one of the etiological factors
   involved in human carcinogenesis in certain regions of the
   world.
  54
Zearalenone
                                                                    Alternariol

Mycotoxins Produced by Alternaria Species

Alternaria has been known for centuries to cause various plant diseases.
Species of this fungus are widely distributed in soil and on aerial plant parts.
More than 20 species of Alternaria are known to produce about 70 secondary
metabolites belonging to a diverse chemical group. However, only alternariol
tenuazonic acid, altertoxin-I, alternariol monomethyl ether (AME), altenuene
are common contaminants in consumable items like fruits (apples), vegetables
(tomato), cereals (sorghum, barely, oat), and other plant parts (such as leaves)

The most common species of Alternaria, A. alternata (formerly known as A.
kikuchiana) produces all important Alternaria toxins including the five
mentioned above and tentoxin, alteniusol, alternaric acid, altenusin,
dehydroaltenusin
  55
• Mycotoxins Produced by Other Fungi



• Sporidesmines, a group of hepatotoxins discovered
  in the 1960s. These mycotoxins, causing facial
  eczema in animals, are produced by Pithomyces
  chartarum and Sporidesmium chartarum and are
  very important economically to the sheep industry.



• Slaframine, a significant mycotoxin produced by
  Rhizoctonia leguminicola (in infested legume forage
  crops).
 56
  PREVENTIVE MEASURES
Management of Mycotoxin Contamination

• The economic implications of the mycotoxin problem and its potential
  health threat to humans have clearly created a need to eliminate or at
  least minimize mycotoxin contamination of food and feed.



• While an association between mycotoxin contamination and inadequate
  storage conditions has long been recognized, studies have revealed that
  seeds are contaminated with mycotoxins prior to harvest . Therefore,
  management of mycotoxin contamination in commodities must include
  both pre- and postharvest control measures



  57
• Preharvest Control
• Mycotoxin contamination can be reduced somewhat by using
  of resistant varieties (most effective, but not all are
  successful) and earlier harvest varieties:
           – crop rotation,
           – adequate irrigation,
           – control of insect pests.

•   Significant control of toxin contamination is expected to be
    dependent on a detailed understanding of the:
           – physiological and environmental factors that affect the
             biosynthesis of the toxin,
           – the biology and ecology of the fungus,
           – the parameters of the host plant–fungal interactions.
• Efforts are underway to study these parameters primarily for
  the most agriculturally significant toxins, namely AFs, Fms,
  and TCTCs
    58
• Use of atoxigenic biocompetitive, native A. flavus strains to
  out-compete the toxigenic isolates has been effective in
  significantly reducing preharvest contamination with
  aflatoxin in cotton and peanuts.

• However, the aflatoxin contamination process is so complex
  that a combination of approaches will be required to
  eliminate or even control the preharvest toxin contamination
  problem.




  59
           Mycotoxins and food chain


         Fungal contamination

                                Vegetables        rare accidents (cancer)
             Many accidents
                                                           Man


       Animal
                                 Products
     Production
                                (Animal origin)


     Elimination




60
Postharvest Control

After harvest, crop should not be allowed to over-winter
in the field as well as subjected to birds and insects
damage or mechanical damage. Grains should be
cleaned and dried quickly to less than 10–13%
moisture and stored in a clean area to avoid insect and
rodent infestation.

Postharvest mycotoxin contamination is prevalent in
most tropical countries due to:
  •a hot, wet climate coupled with
  •subadequate methods of harvesting,
           (handling, and storage practices),
   which often lead to severe fungal growth and mycotoxin
  61
   contamination of food and feed.
• Sometimes contaminated food has been diverted to
  animal feed to prevent economic losses and health
  concerns. However, this is not a solution to the
  contamination problem.

• Irradiation has been suggested as a possible means
  of controlling insect and microbial populations in
  stored food, and consequently, reducing the hazard
  of mycotoxin production under these conditions .

• Significant emphasis has been placed on detoxification
  methods to eliminate the toxins from the contaminated lots or
  at least reduce the toxin hazards by bringing down the
  mycotoxin levels under the acceptable limits.
  62
• I. Removal or Elimination of Mycotoxins.

      – Since most of the mycotoxin burden in contaminated commodities
        is localized to a relatively small number or seeds or kernels
        removal of these contaminated seeds/kernels is effective in
        detoxifying the commodity.
• Methods currently used include:
   – (a) physical separation by:
              – identification and removal of damaged seed;
              –  mechanical or electronic sorting;
              – flotation and density separation of damaged or contaminated seed;
              –  physical screening and subsequent removal of damaged kernels by air
                blowing;
              – washing with water
              – use of specific gravity methods
      All these methods have shown some effect for some mycotoxins,
         including DON, FmB, and AFB1
      – (b) removal by filtration and adsorption onto filter pads, clays,
         activated charcoal, etc.,
      – (c) removal of the mycotoxin by solvent extraction
 63
II. Inactivation of Mycotoxins.
When removal or elimination of mycotoxins is not
possible, mycotoxins can be inactivated by:
 (a) physical methods such as thermal inactivation, photochemical or gamma
irradiation,

(b) chemical methods such a treatment of commodities with acids, alkalies,
aldehydes, oxidizing agents, and gases like chlorine, sulfur dioxide, NaNO2, ozone
and ammonia,

(c) biological methods such as fermentations and enzymatic digestion that cause
the breakdown of mycotoxins. The commercial application of some of these
detoxifying mechanisms is not feasible because, in a number of cases, the methods
will be limited by factors such as the toxicity of the detoxifying agent, nutritional or
aesthetic losses of commodities during treatment, and the cost of the sophisticated
treatment.


Although several detoxification methods have been established for aflatoxins, only
the ammoniation process is an effective and practical method. Other chemicals such
as ozone, chlorine, and bisulfite have been tested and some effect for some
mycotoxins was shown in it. Solvent extractions have been shown to be effective but
are not economically feasible.
 64
  III. Removal of Mycotoxins During Food
  Processing.

• While cooking generally does not destroy
  mycotoxins, some mycotoxins can be detoxified
  or removed by certain kinds of food processing.

   – For example, extrusion cooking appears to be
     effective for detoxifying DON but not AFB.
     FmB1 can form Schiff’s bases with reducing
     sugars such as fructose under certain
     conditions and lose its hepato-carcinogenicity;
     but the hydrolyzed FmB1 was found to be still
  65 toxic.
• Avoiding Human Exposure
Role of Rigorous Monitoring Programs

  While it is impossible to remove mycotoxins completely from foods and
  feeds, effective measures to decrease the risk of exposure depend on a
  rigorous program of monitoring mycotoxins in foods and feeds.
  Consequently, governments in many countries have set limits for
  permissible levels or tolerance levels for a number of mycotoxins in foods
  and feeds.

  Over 50 countries of the world have developed such guidelines. For
  example, levels varying from zero tolerance to 50 ppb have been set for
  total AFs.

  A tolerance level of 1 ppm for DON in grains for human consumption has
  been set by a number of countries, including the United States. The FmB1
  levels established by FDA in 2000 are limited to 5, 20, 60 100, 30, and 10
  ppm, in corn and corn by-products to be used for horse and rabbit, catfish
  and swine, and mink, poultry,

  66
• Among 77 countries which have regulations for
  different mycotoxins, eight have specific regulations
  for OA, with limits ranging from 1 to 20 mg/kg in
  different foods.

• Regulatory guidelines to limit the presence of PT to
  50mg/kg in various foods and juices have been
  established by at least ten countries worldwide.
  Details on worldwide regulatory issues and
  permissive levels of mycotoxins in foods and feeds
  have appeared in a number of recent reviews.



 67
• Detection and Screening of Mycotoxins
•   Because of the diverse chemical structures of mycotoxins, the presence of trace
    amounts of toxins in very complicated matrices that interfere with analysis, and
    the uneven distribution of the toxins in the sample, analysis of mycotoxins is a
    difficult task.

    Because many steps are involved in the analysis, it is not uncommon that the
•   analytical error can amount to 20–30%

•   To obtain reliable analytical data, an adequate sampling program and an
    accurate analytical method are both important.

•   To minimize the errors, studies have led to many improved and innovative
    analytical methods for mycotoxin analysis over the years.

•   New, more sensitive TLC, HPLC, and GC techniques are now available.

•   The MS methods have also been incorporated into HPLC systems.

•   New chemical methods, including capillary electrophoresis and biosensors are
    emerging and have gained application for mycotoxin analysis.

    68
•    After a number of years of research, immunoassays have
    gained wide acceptance as analytical tools for mycotoxins in
    the last decade. Antibodies against almost all the mycotoxins
    are now available. Some quantitative and qualitative
    immunoassays have been approved. Many
    immunoscreening kits, which require less than 15 min. per
    test, are commercially available.
•
    Rather than analysis of toxin, PCR methods, based on the
    primers of key enzymes involved in the biosynthesis of
    mycotoxins, have been introduced for the determination of
    toxicogenic fungi present in foods.

• Detailed protocols for mycotoxin analysis can be seen in
  several of the most recent reviews and books and the most
  recent edition of AOAC .
    69
• Dietary Modifications
• Dietary modification greatly affects the absorption, distribution, and
  metabolism of mycotoxin and subsequently affect its toxicity. For
  example, the carcinogenic effect of AFB1 is affected by nutritional
  factors, dietary additives, and anticarcinogenic substances. Diet
  containing chemoprotective agents and antioxidants such as ascorbic
  acid, and even green tea, have also been found to inhibit carcinogenesis
  caused by AFB1 in test animals.

•   The toxic effect of OA and FmB to test animals was minimized when antioxidants
    such as vitamins C and E are added to the diet. Ascorbic acid also provided
    protective effect against AFs.


• Most mycotoxins have a high affinity for hydrated sodium calcium
  aluminasilicate (HSCAS) and other related products.


    70
 CONCLUSIONS
• Mycotoxins are low molecular weight secondary
  metabolites of fungi that are contaminants of agricultural
  commodities, foods, and feeds.

• Fungi that produce these toxins do so both prior to
  harvest and during storage. Although contamination of
  commodities by toxigenic fungi occurs frequently in
  areas with a hot and humid climate, they can also be
  found in temperate conditions.

• Production of mycotoxins is dependent upon the type of
  producing fungus and environmental conditions such as
  the substrate, water activity (moisture and relative
  humidity), duration of exposure to stress conditions, and
  microbial, insect, or other animal interactions.
 71
• Although outbreaks of mycotoxicoses in humans have been
  documented, several of these have not been well
  characterized, neither has a direct correlation between the
  mycotoxin and resulting toxic effect been well established in
  vivo.


• Even though the specific modes of action of most of the
  toxins are not well established, acute and chronic effects in
  prokaryotic and eukaryotic systems, including humans have
  been reported.


• The toxicity of the mycotoxins varies considerably with the
  toxin, the animal species exposed to it, and the extent of
  exposure, age, and nutritional status.
  72
• Most of the toxic effects of mycotoxins are limited to specific
  organs, but several mycotoxins affect many organs. Induction
  of cancer by some mycotoxins is a major concern as a chronic
  effect of these toxins.


• It is nearly impossible to eliminate mycotoxins from food and
  feed in spite of the regulatory efforts at the national and
  international levels to remove the contaminated commodities.
  This is because mycotoxins are highly stable compounds, the
  producing fungi are ubiquitous, and food contamination can
  occur both before and after harvest. Nevertheless, good farm
  management practices and adequate storage facilities
  minimize the toxin contamination problems.


   73
• A combination of natural biocontrol competition fungi and
  enhancement of host-resistance against fungal growth or toxin
  production could prevent toxin formation to a very significant
  extent.



• Rigorous programs for reducing the risk of human and animal
  exposure to contaminated food and feed also include:

        • economically feasible
        • safe detoxification processes
        • dietary modifications.

   74
• Additional, systematic epidemiological data for
  human exposure is needed for establishing
  toxicological parameters for mycotoxins and the
  safe dose for humans.

• It is unreasonable to expect complete elimination of
  the mycotoxin problem. But multiple approaches will
  be needed to minimize the negative economic
  impact of the toxins on the entire agriculture industry
  as well as their harmful effects on human and
  animal health.


 75

				
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