**4. Trichothecenes**

Trichothecin was first isolated from *Trichothecium roseum* and described by Freeman and Morrison in 1949. The discovery of trichothecin was followed by the isolation and description of other trichothecenes (TCTs), such as diacetoxyscirpenol (DAS), T-2 toxin (T-2), nivalenol (NIV) and deoxynivalenol (DON) (Yazar & Omurtag, 2008).

The Alimentary Toxic Aleukia (ATA) that occurred in Russia during World War II was caused by T-2 toxin and its derivatives; *F. sporotrichioides* was isolated from contaminated grains (Yazar & Omurtag, 2008). DON is the most prevalent toxin associated with Fusarium Head Blight (FHB), and it belongs to the phytotoxic type B trichothecene (Foroud & Eudes, 2009).

TCTs are the most important group of mycotoxins and they are produced above all by various *Fusarium* plant pathogen species (Kimura et al., 2007). They are non-volatile, low-

infected grain, and the predominant pathogens are *F. graminearum* and *F. culmorum*. During infection, *F. graminearum* produces various mycotoxins in grains, in particular deoxynivalenol (DON), a type B trichothecene. *F. graminearum* is the most important DON producer, followed by *Fusarium culmorum*, but other species such as *Fusarium sporotrichioides* or *Fusarium langsethiae* have also been reported. The geographical distribution of the species is probably related to temperature requirements (Merhej et al., 2011). From an economic point of view, the most important TCT producers are *Fusarium* species that cause Fusarium Head Blight (FHB) in small-grain cereals and Gibberella Ear Rot (GER) in maize (Bottalico & Perrone, 2002). The first documented FHB-outbreak occurred in England in 1884, where the disease was named "wheat scab". Outbreaks have since been reported in the Americas, Asia, Australia, Europe, and South Africa (Foroud & Eudes, 2009). These diseases are associated with the temperature in the grain growing region: *F. graminearum* (optimal growth range between 24 and 26°C, minimum aw value 0.90) is more dominant in warmer regions (North America and China), while *F. culmorum* (psychrotrophic strain, optimal temperature growth 21°C) is more dominant in cooler regions (northern Europe) (Sweeney

175

The main species responsible for the production of T-2 toxin is *F. sporotrichioides.* The natural occurrence of this species has been reported in Asia, Africa, South America, Europe and

Apart from *Fusarium*, several other fungal genera are capable of producing TCTs: *Myrothecium*, *Phomopsis, Stachybotrys*, *Trichoderma*, *Trichothecium*. Macrocyclic TCTs are produced largely by *Myrothecium*, *Stachybotrys* and *Trichothecium* (Bennett & Klich, 2003). *Fusarium* species are pathogens that are found on cereal crops and they produce mycotoxins before, or immediately after, harvesting. Consequently, strategies for the prevention of TCTs from entering human and animal food chain include the elimination of TCTs in grains in the field, detoxification of TCTs that are already present in food and feeds, and inhibition of

To date, the control of *Fusarium* proliferation in the field is not ensured, therefore, generation of resistant varieties of crop plants still remain the best way to reduce grain contamination by *Fusarium* without using chemical fungicides. The discovery of biological or chemical molecules which would be able to specifically block the biosynthetic pathway in

At harvest and during the storage of cereals, the key factor for TCT formation is the presence of conidia and humidity combined with temperature. Minimizing or avoiding conidia contaminated materials, cleaning at an early stage during the harvest and drying the grain at low temperatures will allow cereals to be stored for more than 12 months without

TCTs are mainly associated with cereals grown in the temperate regions of Europe, America and Asia: wheat, barley, oats, rye, maize and rice (Yazar & Omurtag, 2008). Their presence has also been reported in soybeans, potatoes, sunflower seeds, peanuts and bananas. TCTs have also been found in processed foods, especially those produced from cereals (bread, breakfast cereals, noodles, and beer). The TCTs that are dominant in grains are deoxynivalenol (DON), nivalenol, and their acetylated derivatives (Foroud & Eudes 2009; Karlovsky, 2011). Corn, wheat, barley, oats, rice, rye and other crops have been reported to

order to limit the residual synthesis of toxins is a new challenge (Merhej et al., 2011).

TCTs absorption in the gastrointestinal tract (He et al., 2010).

increasing TCTs levels (Yazar & Omurtag, 2008).

& Dobson, 1998).

**4.2 Food** 

North America (CAST, 2003).

molecular-weight tricyclic sesquiterpenes with a basic 12,13-epoxy-trichothec-9-ene ring system (Figure 3) and are further classified as macrocyclic, or non macrocyclic depending on the presence of a macrocyclic ester or an ester–ether bridge between C-4 and C-15 (Bennett & Klich, 2003; Merhej et al., 2011). Trichothecenes are a family of more than 200 related compounds which are divided into four subclasses (Types A–D), according to their characteristic functional groups. Type A has a functional group other than a ketone at position C-8; Type B has a ketone at position C-8; Type C has a second epoxy group at C-7,8 or C-9,10 and Type D contains a macrocyclic ring between C-4 and C-5 with two ester linkages (Sweeney & Dobson, 1998). The major Type A trichothecenes in *Fusarium* species include T-2 toxin (T-2) and HT-2 toxin (HT-2), both of which have an isovalerate function in C-8. Type B TCTs include Fusarenone-X, deoxynivalenol (DON) and nivalenol.

Fig. 3. Chemical structure of trichothecens.

The trichothecene biosynthetic pathway in *Fusarium* has been reported extensively by Sweeney & Dobson (1998) and Desjardins & Proctor (2007); it begins with the cyclization of the isoprenioid intermediate farnesyl pyrophosphate to trichodiene by the enzyme trichodiene synthase. After this a number of oxygenation, isomeritation, cyclization, and esterification leading from trichodiene to dyacetoxyscirpenol, T-2 toxin and 3 acetyldeoxynivalenol (Huffman et al., 2010). The recent advances concerning the regulation of trichothecene biosynthesis in *Fusarium* and the potential implication of various general regulatory circuits has been reported in the work of Merhej et al. (2011); the knowledge of the role of these regulatory systems might be useful in designing new strategies to reduce mycotoxin accumulation.

Deoxynivalenol (DON) is the most studied mycotoxin produced by *Fusarium*. DON, also known as vomitotoxin, is a polar organic compound, which is soluble in water and polar organic solvents (e.g. aqueous methanol, ethanol, chloroform, acetonitrile and ethyl acetate); it is optically active. The chemical name of DON is 12,13-epoxy-3α,7α,15 trihydroxytrichothec-9-en-8-one, and the molar mass is 296,32 g mol-1. DON is very stable at temperatures within the 170°C to 350°C interval with no reduction in DON concentration after 30 min at 170°C (Sobrova et al., 2010). DON shows great stability during storage/milling and in the processing and cooking of food.

#### **4.1 Fungi**

The *Fusarium* genus includes a number of important plant pathogens that produce a wide range of mycotoxins (TCTs, fumonisine and zearalenone) which are mainly found in cereal grains (Vesonder & Golinski, 1989). *Fusarium* is the main genus implicated in the production of the non-macrocyclic TCTs. Many toxigenic *Fusarium* species have been associated with

molecular-weight tricyclic sesquiterpenes with a basic 12,13-epoxy-trichothec-9-ene ring system (Figure 3) and are further classified as macrocyclic, or non macrocyclic depending on the presence of a macrocyclic ester or an ester–ether bridge between C-4 and C-15 (Bennett & Klich, 2003; Merhej et al., 2011). Trichothecenes are a family of more than 200 related compounds which are divided into four subclasses (Types A–D), according to their characteristic functional groups. Type A has a functional group other than a ketone at position C-8; Type B has a ketone at position C-8; Type C has a second epoxy group at C-7,8 or C-9,10 and Type D contains a macrocyclic ring between C-4 and C-5 with two ester linkages (Sweeney & Dobson, 1998). The major Type A trichothecenes in *Fusarium* species include T-2 toxin (T-2) and HT-2 toxin (HT-2), both of which have an isovalerate function in

> Trichothecene R1 R2 R3 R4 Deoxynivalenol (DON) -OH -H -OH -OH Nivalenol (NIV) -OH -OH -OH -OH

O

CH3

R2

H H

R1

H

The trichothecene biosynthetic pathway in *Fusarium* has been reported extensively by Sweeney & Dobson (1998) and Desjardins & Proctor (2007); it begins with the cyclization of the isoprenioid intermediate farnesyl pyrophosphate to trichodiene by the enzyme trichodiene synthase. After this a number of oxygenation, isomeritation, cyclization, and esterification leading from trichodiene to dyacetoxyscirpenol, T-2 toxin and 3 acetyldeoxynivalenol (Huffman et al., 2010). The recent advances concerning the regulation of trichothecene biosynthesis in *Fusarium* and the potential implication of various general regulatory circuits has been reported in the work of Merhej et al. (2011); the knowledge of the role of these regulatory systems might be useful in designing new strategies to reduce

Deoxynivalenol (DON) is the most studied mycotoxin produced by *Fusarium*. DON, also known as vomitotoxin, is a polar organic compound, which is soluble in water and polar organic solvents (e.g. aqueous methanol, ethanol, chloroform, acetonitrile and ethyl acetate); it is optically active. The chemical name of DON is 12,13-epoxy-3α,7α,15 trihydroxytrichothec-9-en-8-one, and the molar mass is 296,32 g mol-1. DON is very stable at temperatures within the 170°C to 350°C interval with no reduction in DON concentration after 30 min at 170°C (Sobrova et al., 2010). DON shows great stability during

The *Fusarium* genus includes a number of important plant pathogens that produce a wide range of mycotoxins (TCTs, fumonisine and zearalenone) which are mainly found in cereal grains (Vesonder & Golinski, 1989). *Fusarium* is the main genus implicated in the production of the non-macrocyclic TCTs. Many toxigenic *Fusarium* species have been associated with

C-8. Type B TCTs include Fusarenone-X, deoxynivalenol (DON) and nivalenol.

H3C O

H

CH2 R3

R4

O

Fig. 3. Chemical structure of trichothecens.

storage/milling and in the processing and cooking of food.

mycotoxin accumulation.

**4.1 Fungi** 

infected grain, and the predominant pathogens are *F. graminearum* and *F. culmorum*. During infection, *F. graminearum* produces various mycotoxins in grains, in particular deoxynivalenol (DON), a type B trichothecene. *F. graminearum* is the most important DON producer, followed by *Fusarium culmorum*, but other species such as *Fusarium sporotrichioides* or *Fusarium langsethiae* have also been reported. The geographical distribution of the species is probably related to temperature requirements (Merhej et al., 2011). From an economic point of view, the most important TCT producers are *Fusarium* species that cause Fusarium Head Blight (FHB) in small-grain cereals and Gibberella Ear Rot (GER) in maize (Bottalico & Perrone, 2002). The first documented FHB-outbreak occurred in England in 1884, where the disease was named "wheat scab". Outbreaks have since been reported in the Americas, Asia, Australia, Europe, and South Africa (Foroud & Eudes, 2009). These diseases are associated with the temperature in the grain growing region: *F. graminearum* (optimal growth range between 24 and 26°C, minimum aw value 0.90) is more dominant in warmer regions (North America and China), while *F. culmorum* (psychrotrophic strain, optimal temperature growth 21°C) is more dominant in cooler regions (northern Europe) (Sweeney & Dobson, 1998).

The main species responsible for the production of T-2 toxin is *F. sporotrichioides.* The natural occurrence of this species has been reported in Asia, Africa, South America, Europe and North America (CAST, 2003).

Apart from *Fusarium*, several other fungal genera are capable of producing TCTs: *Myrothecium*, *Phomopsis, Stachybotrys*, *Trichoderma*, *Trichothecium*. Macrocyclic TCTs are produced largely by *Myrothecium*, *Stachybotrys* and *Trichothecium* (Bennett & Klich, 2003).

*Fusarium* species are pathogens that are found on cereal crops and they produce mycotoxins before, or immediately after, harvesting. Consequently, strategies for the prevention of TCTs from entering human and animal food chain include the elimination of TCTs in grains in the field, detoxification of TCTs that are already present in food and feeds, and inhibition of TCTs absorption in the gastrointestinal tract (He et al., 2010).

To date, the control of *Fusarium* proliferation in the field is not ensured, therefore, generation of resistant varieties of crop plants still remain the best way to reduce grain contamination by *Fusarium* without using chemical fungicides. The discovery of biological or chemical molecules which would be able to specifically block the biosynthetic pathway in order to limit the residual synthesis of toxins is a new challenge (Merhej et al., 2011).

At harvest and during the storage of cereals, the key factor for TCT formation is the presence of conidia and humidity combined with temperature. Minimizing or avoiding conidia contaminated materials, cleaning at an early stage during the harvest and drying the grain at low temperatures will allow cereals to be stored for more than 12 months without increasing TCTs levels (Yazar & Omurtag, 2008).

#### **4.2 Food**

TCTs are mainly associated with cereals grown in the temperate regions of Europe, America and Asia: wheat, barley, oats, rye, maize and rice (Yazar & Omurtag, 2008). Their presence has also been reported in soybeans, potatoes, sunflower seeds, peanuts and bananas. TCTs have also been found in processed foods, especially those produced from cereals (bread, breakfast cereals, noodles, and beer). The TCTs that are dominant in grains are deoxynivalenol (DON), nivalenol, and their acetylated derivatives (Foroud & Eudes 2009; Karlovsky, 2011). Corn, wheat, barley, oats, rice, rye and other crops have been reported to

Karlovsky (2011). Bacterial enzymes that catalyze oxidation, epimerization, and, but to a lesser extent, de-epoxidation of DON as well as of the application of acetylation in plant

Fumonisins are a group of non-fluorescent mycotoxins. They were discovered and characterized in 1988 (Bezuidenhout et al., 1988). The predominant fungus isolated from fumonisin contaminated corn, associated with the outbreak of Equine Leukoencephalomalacia (ELEM) in South Africa in 1970 and Porcine Pulmonary Edema (PPE) in Iowa, Illinois, and Georgia in 1989, was *F. verticillioides* (Yazar & Omurtag, 2008). To date, twenty-eight fumonisins have been isolated and they can be divided into four series (A, B, C and P). FB1, FB2 and FB3 are the principal fumonisins analyzed as natural contaminants of cereals (CAST, 2003; Yazar & Omurtag, 2008). Fumonisin B1 is generally the most abundant member of this mycotoxin family; it comprises about 70 % of the total fumonisin content of *Fusarium* cultures (Reddy et al., 2010). Fumonisins have a similar structure to sphingosine, which forms the backbone of sphingolipids within the cell

Fumonisins are polyketide metabolites, derived from the repetitive condensation of acetate units or other short carboxylic acids, via a similar enzymatic mechanism to that responsible for fatty acid synthesis (Huffman et al., 2010). The fumonisin biosynthetic pathway in *Fusarium* species begins with the formation of a linear dimethylatedpolyketide and condensation of the polyketide with alanine, followed by a carbonyl reduction, oxygenations, and esterification with two propane-1,2,3-tricarboxylic acids (Desjardins &

Fumonisin biosynthetic genes have been mapped to one locus in the *F. verticillioides* genome

The basic chemical structure of fumonisins is a C-20 aliphatic chain with two ester linked hydrophilic side chains (Richard, 2007). The chemical structure of FB1 is 1,2,3- Propanetricarboxylic acid, 1,1N-[1- (12 amino-4,9,11-trihydroxy-2-methyltridecyl)- 2-(1 methylpentyl)-1,2-ethanediyl] Ester (Figure 4). FB2 is the C-10-deoxy analogue of FB1 and FB3 is the C-5-deoxy analogue of FB1(Yazar & Omurtag, 2008). The molecular mass of FB1 is 721 g/mol, while FB2 and FB3 have the same value of molecular mass (705g/mol). FB1 is soluble in water to at least to 20 mg/ml, and in methanol and acetonitrile-water. FB1 and

OH

NH2

177

OH OH

COOH

O

O

O

O COOH

COOH

HOOC

biotechnology have been described (He et al., 2010).

membrane (Sweeney & Dobson, 1998).

**5. Fumonisins** 

Proctor, 2007).

(Desjardins & Proctor, 2007).

Fig. 4. Chemical structure of fumonisin B1.

contain T-2 toxin (CAST, 2003). Moreover, TCTs can enter the food chain through milk, meat and eggs from livestock and poultry that are fed with contaminated feeds, although the exposure risk to human through the consumption of animal tissue is much less than the direct consumption of contaminated grains (He et al., 2010).

Food and feed contamination by TCT have been associated with chronic and fatal toxicoses of humans and animals, including Alimentary Toxic Aleukia in Russia and Central Asia, Akakabi-byo (red mould disease) in Japan, and swine feed refusal in the central United States (Karlovsky, 2011). The epidemy that occurred in Russia between 1942 and 1948, where at least 100,000 people died, was caused by the ingestion of grain contaminated with T-2 produced by *F. sporotrichoides* or *F. poae* (Foroud & Eudes, 2009) .

### **4.3 Toxicity**

At the cellular level, the main mechanism of TCT mycotoxins appears to be a primary inhibition of ribosomal protein synthesis, which is followed by a secondary disruption of DNA and RNA synthesis (Desjardins & Proctor, 2007; Richard, 2007; Zain, 2011), cytotoxicity, and apoptosis (Rocha et al., 2005; Rotter et al., 1996).

TCTs affect dividing cells, such as those coating the gastrointestinal tract, the skin, and lymphoid and erythroid cells. The toxic action of TCTs results in extensive necrosis of the oral mucous and skin in contact with the toxin, an acute effect on the digestive tract and decreased bone marrow and immune functions (Richard, 2007; Rocha et al., 2005).

In general, acute exposure of animals to DON resultes in decreased feed consumption (anorexia) and vomiting (emesis), while longer exposure causes reduced growth, and adverse effects on the thymus, spleen, heart, and liver (Sobrova et al., 2010).

Nowadays, the real concern is not related to acute exposure, but to a prolonged daily exposure, which leads to chronic toxicity, since it has been demonstrated that DON deregulates the immune response and induces cytokine up regulation (Merhej et al., 2011; Pestka & Smolinskj, 2005). It has been demonstrated that the ingestion of DON with contaminated feeds and food leads to growth retardation, and reproductive disorders in animals (Pestka, 2010; Rocha et al., 2005; Sobrova et al., 2010). To date, all the animal species evaluated have shown a differential level of susceptibility to DON with the pigs being the most susceptible (Pestka & Smolinski, 2005).

Human exposure to DON-contaminated grains has been reported to cause acute temporary nausea, vomiting, diarrhea, abdominal pain, headache, dizziness, and fever (Sobrova et al., 2010).

In general, TCTs are heat-stable molecules and are not fully eliminated during the processes currently used in cereal-based food manufacturing (Hazel & Patel 2004). They are also stable at neutral and acidic pH and consequently, they are not hydrolyzed in the stomach after ingestion (Yazar & Omurtag 2008). Since DON is water soluble, its level is reduced in cooked pasta (Sobrova et al., 2010).

The chemical detoxification of DON by ozone (Young et al., 2006), ammonia, chlorine, hydrogen peroxide (He et al., 2010), sodium bisulfite (Young et al. 1986), sodium carbonate (Abramson et al. 2005), and chlorine dioxide (Wilson et al., 2005) has been demonstrated. Therefore, the best way to prevent contamination would be to limit TCT biosynthesis at the field level during crop cultivation (Merhej et al., 2011).

The enzymes involved in biological detoxification of DON and their application to genetically engineered crops and feed additives have been reviewed in the work by

contain T-2 toxin (CAST, 2003). Moreover, TCTs can enter the food chain through milk, meat and eggs from livestock and poultry that are fed with contaminated feeds, although the exposure risk to human through the consumption of animal tissue is much less than the

Food and feed contamination by TCT have been associated with chronic and fatal toxicoses of humans and animals, including Alimentary Toxic Aleukia in Russia and Central Asia, Akakabi-byo (red mould disease) in Japan, and swine feed refusal in the central United States (Karlovsky, 2011). The epidemy that occurred in Russia between 1942 and 1948, where at least 100,000 people died, was caused by the ingestion of grain contaminated with

At the cellular level, the main mechanism of TCT mycotoxins appears to be a primary inhibition of ribosomal protein synthesis, which is followed by a secondary disruption of DNA and RNA synthesis (Desjardins & Proctor, 2007; Richard, 2007; Zain, 2011),

TCTs affect dividing cells, such as those coating the gastrointestinal tract, the skin, and lymphoid and erythroid cells. The toxic action of TCTs results in extensive necrosis of the oral mucous and skin in contact with the toxin, an acute effect on the digestive tract and

In general, acute exposure of animals to DON resultes in decreased feed consumption (anorexia) and vomiting (emesis), while longer exposure causes reduced growth, and

Nowadays, the real concern is not related to acute exposure, but to a prolonged daily exposure, which leads to chronic toxicity, since it has been demonstrated that DON deregulates the immune response and induces cytokine up regulation (Merhej et al., 2011; Pestka & Smolinskj, 2005). It has been demonstrated that the ingestion of DON with contaminated feeds and food leads to growth retardation, and reproductive disorders in animals (Pestka, 2010; Rocha et al., 2005; Sobrova et al., 2010). To date, all the animal species evaluated have shown a differential level of susceptibility to DON with the pigs being the

Human exposure to DON-contaminated grains has been reported to cause acute temporary nausea, vomiting, diarrhea, abdominal pain, headache, dizziness, and fever (Sobrova et al.,

In general, TCTs are heat-stable molecules and are not fully eliminated during the processes currently used in cereal-based food manufacturing (Hazel & Patel 2004). They are also stable at neutral and acidic pH and consequently, they are not hydrolyzed in the stomach after ingestion (Yazar & Omurtag 2008). Since DON is water soluble, its level is reduced in

The chemical detoxification of DON by ozone (Young et al., 2006), ammonia, chlorine, hydrogen peroxide (He et al., 2010), sodium bisulfite (Young et al. 1986), sodium carbonate (Abramson et al. 2005), and chlorine dioxide (Wilson et al., 2005) has been demonstrated. Therefore, the best way to prevent contamination would be to limit TCT biosynthesis at the

The enzymes involved in biological detoxification of DON and their application to genetically engineered crops and feed additives have been reviewed in the work by

decreased bone marrow and immune functions (Richard, 2007; Rocha et al., 2005).

adverse effects on the thymus, spleen, heart, and liver (Sobrova et al., 2010).

direct consumption of contaminated grains (He et al., 2010).

**4.3 Toxicity** 

2010).

T-2 produced by *F. sporotrichoides* or *F. poae* (Foroud & Eudes, 2009) .

cytotoxicity, and apoptosis (Rocha et al., 2005; Rotter et al., 1996).

most susceptible (Pestka & Smolinski, 2005).

cooked pasta (Sobrova et al., 2010).

field level during crop cultivation (Merhej et al., 2011).

Karlovsky (2011). Bacterial enzymes that catalyze oxidation, epimerization, and, but to a lesser extent, de-epoxidation of DON as well as of the application of acetylation in plant biotechnology have been described (He et al., 2010).
