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MYCOTOXINS

• Produced by fungi belonging to different genera (Aspergillus, Fusarium, Penicillum..)

• Secondary metabolites:

– Compounds not necessary for the life and the growth of the fungi but it is synthetized during the stationary phase of its life cycle

– Uncertain physiological role

– For mycotoxins, some roles have been hypothesized

• Their production depends on specific environmental conditions (not dependent on their growth!)

MYCOTOXINS

• Represent a group of chemicals that can occur in a variety of plants used as food

– Cereal grains (barley, maize, rye, wheat)

– Coffee

– Dried fruits (nuts, peanuts)

– Spices

• Some may occur in animal products derived from animals that consumed contaminated feed

MYCOTOXINS

• Extremely stable and non-eliminable products

– Thermostable, colorless, odorless

• Able to contaminate both vegetable and animal matrices

• If accumulated into animals, can be excreted with animal secondary products used as human food

– Entrance in the human food chain

MYCOTOXINS

Fungal contamination

Vegetables

Man

Animal

Production Products

(Animal origin)

Many accidents

rare accidents

Elimination

MYCOTOXINS PRODUCING FUNGI

• Mild parasite:

– Not aggressive, colonizing the host through already existing damages

(i.e. insects)

• Sensitive to environmental conditions:

– Various factors (i.e. methods and conditions of cultures, climate changes)

• Mycotoxins production induced by stress conditions:

– Possible defense role?

MYCOTOXINS PRODUCING FUNGI

• FIELD FUNGI:

– fungi that attack plants that grow in the field (occurring prior to harvest) grow under special conditions (Aspergillum and Fusarium)

• STORAGE FUNGI:

– Storage fungi usually invade grain or seed during storage and are generally not present in large quantities before harvest in the field (Penicillium). Contamination occurs through spores contaminating the grain as it is going into storage from the harvest.

PROMOTING FACTORS FOR MYCOTOXINS PRODUCTION

• PRE-HARVEST FACTORS

• Biological factors:

– Presence of fungi (but it is not a rule)

– Sensitivity of the plant (polygenetic and epigenetic resistance)

• Environmental factors:

– Temperature

– Humidity/rain

• Control of adverse factors

– Weeding (reduction of stress induced by plant competition)

– Parasites control

• POST-HARVEST FACTORS

• Harvest phase

– Phenologic phase

– Temperature during harvesting

– Humidity during harvesting

– Time before manufacturing and/or storage


• POST-HARVEST FACTORS

• Storage phase

– Humidity

– Temperature

– Gas composition in the surrounding atmosphere

– Biological factors (i.e. insects)

• Manufacturing phase

– sorting

– transformations


MYCOTOXINS

• So far, almost 400 different mycotoxins have been identified

• Progress in the analytical detection methods is increasing novel mycotoxins identification (i.e. masked mycotoxins)

• Among these, almost 10% is the toxic ones

• Concerning FOOD SAFETY, warning is given for 10-20 mycotoxins.

THE MAIN MYCOTOXINS

Aflatoxins

Fumonisins

Ochratoxin A

Deoxynivalenol

Zearalenone

Aspergillus flavus

Aspergillus parassiticus

Fusarium verticilloides

Fusarium proliferatum

Aspergillus ochraceus

Penicillium verrucosum

Fusarium graminearum

Fusarium culmorum

Maize, dried fruits, oilseeds, wheat..

Maize, maize products..

Grain, raisin, coffee..

Grain

MYCOTOXINS ASSOCIATED WITH «FOOD» AND «FEED»

• Aflatoxins (B1, B2, G1, G2, M1)

• Ochratoxin A

• Zearalenone

• Fumonisins

• Trichothecenes (Deoxynivalenol)

• Ergot alkaloids

• Patulin

• Moniliform

• Sterigmatocystin

• Citrinin

• Cyclopiazonic acid

• Kojic acid

• Maltoryzine

• ß-nitropropionic acid

• Aspergillic acid

• Penicillic acid

• Roquefortine C

CONTAMINABLE RAW MATERIALS

• GRAIN: wheat, maize, barley, rye, oat..

• OILSEEDS: sunflower, cotton seeds..

• FRUITS and VEGETABLES: grapes, apples, pears, carrots, tomatoes..

• DRIED FRUITS: nut, peanuts, almonds, hazelnut, dried fig..

• COFFEE

• CACAO

• SPICES: pepper, chili pepper, ginger..

• MILK and DERIVATIVES: milk, cheese, yogurt

• EGGS

• MEAT

CONTAMINABLE RAW MATERIALS

MYCOTOXICOSIS

• MYCOTOXICOSIS: the action of mycotoxins on different organs (i.e. liver, kidney, lungs) and systems (i.e. nervous, endocrine, and immune systems).

DO NOT CONFUSE WITH

• MYCOSIS: a generalized invasion of living tissues by growing fungi.

MICOTOXICOSIS: HUMAN EFFECTS

• Differently to bacterial toxins, mycotoxins are not protein compounds and therefore are not detected by the immune system

• Different symptoms, depending on the mycotoxin (different mechanisms of action)

• Some toxins induce very mild effects, others induce severe symptoms (death)

• Human severe adverse effects:

– Genotoxicity, cancerogenesis, mutagenesis, teratogenesis, nephrotoxicity, immunotoxicity, dermatotoxicity

MICOTOXICOSIS: TOXICITY

• Acute exposure (high doses)

– Mainly in underdeveloped countries

– Es: Ergotism (Etiopia) and Aflatoxicosis (Kenya)

• Chronic exposure (doses > TDI)

– Cancerogenesis

– Growth delay

– Immunosoppressions

• Chronic exposure (doses < TDI)

– Immunosoppressions

MYCOTOXICOSIS

• 1960-1963: a series of case reports that associated the death of turkeys (England, Turkey X Disease) and ducklings (Uganda) with consumption of peanuts feed containing products produced by A. flavus

• 1967 (Taiwan): 26 people in a farming community became ill after eating contaminated rice; 3 children died.

– Cause of death: Contaminated rice with >200 µg aflatoxin B1/kg.

MYCOTOXICOSIS

• 1974 (India): outbreak of hepatitis affected 400 people resulting in 108 deaths.

– Cause of death: aflatoxins in corn ( 6-15 mg/kg)

• 2004 (Kenya): one of the largest aflatoxicosis outbreak resulting in 317 cases and 125 deaths.

– Cause of death: corn contaminated with 4.4 mg/kg of aflatoxin B1, 220 times higher than Kenyan regulatory limit for food.

REGULATORY LIMITS

• First limit for mycotoxins was established in 1965 (FDA)

• Official regulations for mycotoxins are extremely complex and are continuously evolving.

• Official regulations are usually set up both for human food and animal feed with obvious differences

– FOR HUMAN FOOD: Reg. (CE) N. 1881/2006, Reg. (CE) N. 1126/2007 and Reg. (CE) N. 165/2010

– FOR ANIMAL FEED: Directive 2002/32/CE and subsequent upgrades, Recommendation 2006/576/CE and Reg. (CE) N. 574/2011

REGULATORY LIMITS

EU Regulation 1881/2006 Mycotoxin Fruits,

etc

Cereals,

etc

Milk, etc Coffee,

wine

Nuts,

spices

Baby food

Aflatoxins

B1B2G1G2

Aflatoxin M1 Trichothecenes (DON, T2 toxins) Zearalenone Ochratoxin A Patulin

AFLATOXINS • Produced by: A. flavus and A. parassiticus

• Contaminants of: maize, dried fruits, oilseeds

• Promoting factors:

– Temperature: 25-30°C

– Humidity of grain

• Main toxins:

– Aflatoxin B1 and B2, G1 and G2

– Aflatoxin M1 (animal metabolite found in urine, milk, tissues..)

• Poisoning is called AFLATOXICOSIS

AFLATOXINS

HUMAN TOXICITY

• In general, they are genotoxic carcinogens.

• Acute effects: abdominal pain, vomiting, hepatic necrosis, death

• Chronic effects:

– Carcinogenesis: hepatic tumor, additive effects in co-presence with Hepatitis B virus

– Aflatoxins B1: Class 1 «Carcinogenic to humans» (IARC)

– Reduced reproductive and immune functions

– Delayed growth in newborn patients after ingestion of contaminated milk (Aflatoxin M1)

– Aflatoxin M1: Class 2B «Possibly carcinogenic to humans» (IARC)

MECHANISM OF TOXICITY • Metabolized by Cytochrome P450

(CYP3A4) in the liver

• The most toxic metabolite is the aflatoxin 8,9 epoxide that is able to be incorporated into the DNA after forming an adduct with guanine

• Aflatoxin 8,9 epoxide DNA intercalation induces a mutation on p53 gene

• This mutation is pretty resistant to DNA repair processes

• The shut off of the gene induces an uncontrolled cell proliferation that may lead to hepatic carcinoma

MECHANISM OF TOXICITY

• In case of acute exposure, aflatoxins inhibit carbohydrate and lipid metabolism as well as protein synthesis

• Inhibition of protein traduction (< functioning mRNA, < protein synthesis)

• Reduced production of functioning enzymes and «lipid acceptor proteins» that could induce hepatic steatosis

EU LIMITS (Reg. (CE) N. 1881/2006)

• No exposure levels to aflatoxins could be considered as safe for humans

• EU limits more stringent than USA

• In general, EU limit = 10 µg/kg for food used for direct human consumption

• In oilseeds: 2 µg/kg of AFB1 and 4 µg/kg of total aflatoxins (8 and 15 µg/kg in oilseeds for further processing)

• In dried fruits for direct human consumption: 5 µg/kg of AFB1 and 10 µg/kg of total aflatoxins.

• In baby milk/food: < 0.1 µg/kg of AFB1 and 0.05 µg/kg of AFM1.

FUMONISINS

• Produced by: F. verticillioides, F. proliferatum

• Contaminants of: maize and maize products

• Main toxins: Fumonisin B1 and B2

HUMAN TOXICITY

• Toxicity exerted at the hepatic and renal levels

• Fumonisin B1: Class 2B «Possibly carcinogenic to humans» (IARC)

• Fumonisins have been associated also with alterations in the neuronal tube and induction of esophagic tumor


• Inhibition of the synthesis of ceramide (Ceramide synthetase).

• Disruption of sphingolipids metabolism (brain, liver)

• SPHINGOLIPIDS functions:

– Cell-cell comunication

– Regulation of cell surface receptors

– Regulation of intracellular signalling


• Implications of SPHINGOLIPIDS on:

– Cell cylce control

– Apoptosis

– Cell proliferation


• Inhibition of sphingolipids metabolism is based on the structural similarity with fumonisins

• Competitive inhibition


• TDI of 2µg/kg bw/day

• In corn based products: 500 µg/kg

• In cornflakes: 200 µg/kg

• In baby food: 100 µg/kg

OCHRATOXIN A

• Produced by: A. ochraceus, P. cyclopium and P. verrucosum

• Contaminants of: grains, dried fruits, grapes, coffee, chocolate, olive..


– Temperature: 24-25°C

• Main ochratoxins: - Ochratoxin A

- Ochratoxin B - Ochratoxin C

HUMAN TOXICITY

• The entrance in the food chain is associated with the ability to bind to the animal serum albumins.

• Ochratoxin A is the most toxic compound:

– Nephrotoxicity

– Hepatic necrosis

• Acute exposure: vomiting, diarrhea

• Chronic exposure: immunosuppression, teratogenicity, carcinogenesis

– Ochratoxin A: Class 2B «Possibly carcinogenic to humans» (IARC)


• Inhibitor of protein synthesis (false substrate of the enzyme):

– Inhibition of phenylalanine t-RNA synthase

– Inhibition of phenylalanine hydroxylase


• Inhibitor of cellular energy production:

– Inhibition of phosphoenolpyruvate-carboxykinase (interference at the mRNA level)

– Involved in the metabolism of gluconeogenesis

• Genotoxic effects:

– electrophilic products due to ochratoxins bioactivation can covalently bind to DNA causing mutations

– activation by peroxidases as an important step to produce the phenoxyl radical

– parachlorophenol structure can undergo CYP450-catalized oxidative dechlorination, resulting in a quinoidal structure that can bind covalently to thiol groups of nitrogenous bases (especially G)


• TDI < 5 µg/kg/day

• In dried fruits and soluble coffee: 10 µg/kg

• In roasted coffee: 5 µg/kg

• In wine and grape juice: 2 µg/kg

• In beer: 0.2 µg/kg

• In spices: 15-30 µg/kg

TRICHOTHECENS

• Produced by: several species of Fusarium

• Contaminants of: maize, wheat, barley, rice


– High humidity

• Trichothecens are produced by several other species:

– Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium, e Stachybotrys

• Almost 170 different toxins characterized by a sesquiterpenoide tetracyclic ring 12,13-epossitricotecen-9-ene, la cui la tossicità è dovuta al gruppo epossidico

GROUP A: No carbonile group on C8

• T-2 toxin and HT-2 toxin

GROUP B: Carbonile group on C8

• Deoxinivalenol and analogues

HUMAN TOXICITY • Toxicity exerted at different levels:

– Gastrointestinal, cutaneous, hematopoietic, nervous, immunity, hepatic and cardiovascular

– In addition, deoxivalenol displays an anorectic and emetic effects (vomitotoxin).


• Trichothecens inhibit the protein synthesis

• Inhibition of the elongation phase of protein synthesis by altering the peptide-bond formation


• TDI = 1 µg/kg/day

• Deoxyvalenol:

• In grain based products: 500 µg/kg


• T2 and HT2 toxin:



ZEARALENONE

• Produced by: F. graminearum

• Contaminants of: maize, wheat, barley, oat


– Temperature: alternating high and low 7-21°C

– Humidity: 22-25%

HUMAN TOXICITY

• Non steroidal estrogenic activity

– Binding to the receptor for the 17β-estradiol with an activity 2-4 times lower than estradiol.

– Infertility, abortion

– Early puberty


• TDI = 0,2 µg/kg/day



MASKED MYCOTOXINS

• The first was identified in 2004

• In 2011, ILSI EU Expert group on masked mycotoxins released the first harmonized definition of masked mycotoxins:

– MASKED MYCOTOXINS: mycotoxins metabolites produced directly on the plants during their normal life cycle

– MODIFIED MYCOTOXINS: mycotoxins metabolites produced during storage and manufacturing of plants or in secondary organisms (es. animals)

• Very frequently, masked mycotoxins are conjugation products with polar groups: detoxifications of the plant

MASKED MYCOTOXINS • Main mycotoxins: deoxynivalenol and zearalenone

• Main chemical reactions: glucosylation and sulfation

• The major masked mycotoxins are:

– DON-3-glu

– ZEN-4-glu

• Main problems are:

– Very few toxicological data

– Detection methods (crossreactivity, over-estimation)

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