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Mycotoxins

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Introduction

• Mycotoxin is a convenient generic term describing the toxic secondary metabolites produced by fungi.

Mycotoxins are secondary metabolites of fungi that are recognized as toxic to other life forms.“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.

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 cycle. Many are used in foods, pharmaceuticals, and other industrial applications.)

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Some mycotoxins could also be toxic to plants or othermicroorganisms; but these compounds are not classified asantibiotics of fungal origin.

Like most microbial secondary metabolites, the benefit of mycotoxins for the fungi themselves is still not clearly defined.

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.

Introduction

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• 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.

• 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.

Introduction

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• Mycosis” refers to a generalized invasion of living tissue(s) by

growing fungi.

1. Fungal growth

a. Field fungi : grow under conditions occurring prior to harvest. (Fusarium)

b. Storage fungi : do not invade intact grain prior to harvest. (Aspergillus & Penicillium)

Introduction

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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

Introduction

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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

3. Treatment of mycotoxin - induced disease

Introduction

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a. Avoiding

b. Diluting

c. Cleaning

d. Drying

e. Adding (organic acids will prevent mold growth)

4 .Prevention of mycotoxin-induced disease

Introduction

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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:

Aspergilus flavus, Alternaria longipes, Alternaria alternata, Claviceps purpura, Fusarium verticillioides (previously called moniliforme), Fusarium graminearum, and a number of other Fusarium spp.

I- Production of mycotoxins by toxogenic fungi

Species most likely introduced at harvest include:

• Fusarium sporotrichioides, Stachybotrys atra, Cladosporium sp., Myrothecium verrucaria, Trichothecium roseum, as well as Alternaria alternata.

Species most likely found in the storage are mostly from genus Penicillia and 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.

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• 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

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II- 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.

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• 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

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.

• 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.

Water activity or aw was developed to account for the intensity with which water associates with various non-aqueous constituents and solids. Simply stated, it is a measure of the energy status of the water in a system. It is defined as the vapor pressure of a liquid divided by that of

pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one

Continue Factors Affecting…….

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III- The most important Mycotoxins

R1

R2

R3

R4

A B

D C

R1

R1 R1

Chemical Structure of Different Aflatoxins

1 )Aflatoxins

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Sources

Aspergillus flavus & A. paraciticus on Corn and peanuts

Factor favoring production of aflatoxinsa. Grain moistureb. Tempreture

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.

Chemical characteristics

Exhibit intense blue or green fluorescence under UV.

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hepatic steatosis Accumulation of large vacuoles of triglyceride fat in

liver cells via the process of steatosis

Mechanism of toxicological damage

Pathogenesis Aflatoxicosis

Aflatoxin bind to guanine in deoxyribonucleic acid (DNA) inhibiting the signal for the formation of massenger ribonucleic acid (mRNA).

Toxicity

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Interruption of protein synthesis leads to deficiencies of structural protein.

The long term effects of impaired protein synthesis include hepatic steatosis and variety of metabolic and function derangements like:

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

Young animals are more susceptible than adult and the nutrition deficiency increase susceptibility

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• 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.

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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.

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Diagnosis

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

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.

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The Most Important Types of of Aflatoxins

At least 16 structurally related toxins in this group and the most prominent of them are Aflatoxins B1, B2, G1, G2, M1and M2.

They are produced by Asparagillus flavus and A. parasiticus and infrequently A. pseudotamarii and A. nominus A. ochraceoroseus has also been found to produce aflatoxins

Aflatoxin B1 is the 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 animal urine, bovine milk or tissues.

Aflatoxins have been found in corn, peanuts سودانّي� and فولpeanut products, cotton seeds, peppers, rice, pistachios, فستق tree nuts, pumpkin قرع seeds, sunflower seeds and other oil seeds, copra, هند .spices, and dried fruits (figs, raisins) جوز

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.

Natural Occurrence

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• Soybeans الصويا, beans الفاصولياء, pulses (Pea)البازالء, cassava ,القطن oats ,القمح wheat ,الدخن millet , الذرة sorghum ,منيهوتbarley الشعير, and riceرز are 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.

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2 )Ochratoxin & Citrinin

Sources :

Aspergillus orchraceus & Penicillium viridicatum

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

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Clinical sign

a. Acute : vomiting, diarrhea, dehydration & depression

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

The Most Important Types of Fungi

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.

تشوهات جنينية

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• Other fungi, such as Petromyces alliceus, Aspergillus ciricus, and Aspergillus fonsecaeus (both in Aspergillus niger group), have also been found to produce OA.

• Most of the OA producers are storage fungi and pre-harvest fungal infection.

Factors affecting OA

• Although most OA producers can grow in a range from 37Cto 48C 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.

Natural Occurrence• 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.

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• These metabolites form different classes depending on the nature of the amide group, and the presence or absence of a chlorine at R2 in the phenyl group.

Structure of the ochratoxins.

• 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.

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• Ochratoxin A, the most toxic member of this group of mycotoxins, has been found to causing kidney damage as well as liver necrosis and enteritis (Small Intestine) in many animal species.

• The OA inhibits the activity of different enzymes like, carboxypeptidase A, renal phosphoenolpyruvate carboxykinase, phenylalaninetRNA synthetase, and phenylalanine hydroxylase.

• Formation of free radicals has been considered as one of the mechanisms for the carcinogenic/toxic effects of OA.

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3) Fumonisins

• Fumonisins (Fm) are a group of toxic metabolites produced primarily by Fusarium 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.

• 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.

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Toxicological Effects

Fumonisin B1 is primarily a hepatotoxin and carcinogen in rats. Feeding culture material from F. verticillioides or pure FmB1 to rats resulted in cirrhosis and hepatic nodules, carcinoma.

تلي�ف كبدّي�

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.

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Several species of Fusaria infect corn, wheat, barley, and rice.

Under favorable conditions, they elaborate a number of different types of mycotoxins, more than 100 TCTCs have been identified.

Only a few frequently found in foods and feeds are potentially hazardous to human and animal health.

4) Trichothecenes (TCTCs)

Trichothecenes

a) T-2 toxin,b) Deoxynivalenol (DON)

chemical structure of the side chain.

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Other fungal genera elaborate TCTCs are: Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium, and Stachybotrys.

The TCTC mycotoxicoses affect many organs, including the gastrointestinal tract, 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

T-2 toxin, a highly toxic type of A TCTC. It 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.

Almost all the major TCTCs, including T-2 toxin, are cytotoxic and cause hemorrhage, edema, and necrosis of skin tissues.

4-a) T-2 toxin

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4-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.

Worldwide frequent natural occurrence of DON in cereal grains has been reported.

Contamination of this toxin in corn and wheat is generally high.

• 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.

• The optimal temperature for DON production is about 24C.

• 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.

Edema is an abnormal accumulation of fluid beneath the skin Anorexia is the symptom of poor appetiteCytokines are small cell-signaling protein molecules that are secreted by cells of the nervous system

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5 )ZearalenoneSources: Fusarium roseum and F. graminearum on corn, wheat, barley & oats

Factor favoring production

a. High moisture 22% - 25%

b. Alternating high and low temp. (7-21 ๐c)

Mechanism of toxicological damage

a. initiating specific RNA synthesis

b. Function as a weak estrogen.

Toxicitya. Swine are most susceptibleb. low for all effects except reproductive function.

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Source : Claviceps purpurea on barley, wheat & oats

Factor favoring : Warm & humid

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

6) Ergot

Dopamine a simple organic chemicalDopamine plays a major role in the brain system that is responsible for reward-driven learning Several important diseases of the nervous system are associated with dysfunctions of the dopamine system Parkinson's disease

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Treatment

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

b. control secondary bacterial infection

c. milk supplement

Mechanism of toxic

a. potent initiators of contraction in smooth muscle

b. mimic the action of dopamine.

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Mycotoxins and food chain

Fungal contamination

Vegetables

Man

Animal

ProductionProducts

( Animal origin)

Many accidents

rare accidents (cancer)

Elimination

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I- 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 post- harvest control measures

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I-A- Pre-harvest 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

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• 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.

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I-B- Post-harvest 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:

• subadequate methods of harvesting, a hot, wet climate coupled with handling, and storage

practices which often lead to severe fungal growth and mycotoxin contamination of food and feed.

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• 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.

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II- 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 removal by :

(a) filtration and adsorption onto filter pads, clays, activated charcoal, etc.,

(b) solvent extraction removal of the mycotoxin by some specific solvent

(c) 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, AFB1

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III- 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.

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IV- Avoiding Human Exposure

Role of Rigorous Monitoring Programs

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, respectively.

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.

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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.

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• Although occurrences 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.

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• 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.

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• 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.

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• 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.