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Mode of action | Toxicology | Metabolism
Mycotoxins as a group cannot be classified according to their mode of action, toxicology or metabolism. These vary according to the different chemical structures, sensitivity of the species to the toxin and also factors such as sex, age, health and diet. There are a large number of mycotoxins with a great diversity in their mode of action. Additionally, additive and synergistic effects can occur in the presence of two or more mycotoxins. Following there are the most important mycotoxins and their mode of actions, toxicology and metabolism as well as main symptoms and target organs.
Aflatoxins are one of the most studied mycotoxins and were identified in 1960. They are mainly produced by certain strains of Aspergillus parasiticus and A. flavus and they occur in agricultural products in tropical and subtropical regions. Aflatoxins are divided into six major toxins according to their fluorescent properties under ultraviolet light (ca. 365 nm) and their chromatographic mobility (subscripts). Aflatoxins B1 and B2 produce a blue fluorescence while G1 and G2 a green one. There are also two metabolic products aflatoxin M1 and M2 which occur in the milk of lactating mammals which have consumed aflatoxin contaminated feed. Aflatoxin B1 is the most toxic and the most prevalent among this family.
Chemical structure of the different aflatoxins
Exposure and absorption into the organism
Because of aflatoxins’ common occurrence in feedstuffs, feeds and milk products, these mycotoxins pose a serious threat to humans and animal species. Although the oral route is the main contamination means, inhalation may also occur as a result of people or animals being exposed to the grains’ dust. After respiratory exposure, AfB1 may appear in the blood more quickly than after oral exposure. Nevertheless, after 4 hours the plasmatic concentration does not differ between the two routes of contamination. Following ingestion, aflatoxin B1 is efficiently absorbed in the intestinal tract, of which the duodenum appears to be the major site of absorption. Due to the particle’s low molecular weight, the main mechanism of absorption of mycotoxin, as suggested by several authors, is passive diffusion, in which no efflux pumps or transporters are involved.
The main metabolizing organ for aflatoxin is the liver, but this can also occur directly at the site of absorption, in the blood or in several extra-hepatic organs.
The metabolism of AfB1 can be divided into three phases:
In this phase aflatoxins exert their toxic effects. At this first stage, aflatoxin B1 is oxidized into several hydroxylated metabolites.
The metabolic pathways for AfB1 include o-demethylation to AfP1, reduction to aflatoxicol and hydroxylation to AfB1-8,9-epoxide (acutely toxic, mutagenic, and carcinogenic), AfM1 (acutely toxic), AfFQ1, or AfB2 (both relatively non-toxic).
Aflatoxin B1 pathways
modified from Yiannikouris and Jouany, 2002
Aflatoxin B1– 8,9 epoxide is highly unstable, thus several reactions may occur, depending on the second molecule present:
Biological nucleophils (such as nucleic acids) – stable links to RNA and DNA are formed; inducing point mutations and DNA strand breaks. These reactions and the formation of AfB1-DNA adducts are highly correlated with the carcinogenic effect of AfB1 in both animal and human cancer cases.
Water – in the presence of water molecules, Aflatoxin B1– 8,9 epoxide will be hydrolyzed into AfB1– 8,9- dihydrodiol and become available to be linked with serum proteins, such as lysine and albumin. This mechanism may explain the toxic effects of aflatoxin.
Phase I metabolites may undergo phase II biotransformation involving the enzymes glutathione S-transferase (GST), ß-glucuronidase, and/or sulfate transferase which produce conjugates of AfB1-glutathione, AfB1-glucuronide, and AFB1-sulfate, respectively. The major conjugate of AfB1-epoxide identified is the AfB1-glutathione conjugate. This conjugation is the principal detoxification pathway of activated AfB1 in many mammals which is essential in the reduction and prevention of AfB1 induced carcinogenicity. The resulting conjugates are readily excreted via the bile into the intestinal tract. It has been accepted that cytosolic GST activity is inversely correlated to the susceptibility of several animal species to AfB1 carcinogenicity.
This phase can occur in the intestinal tract as a result of bacterial activity. Deconjugation is part of the metabolic role of the large intestine flora, which results in reabsorption and an enterohepatic circulation is established.
Excretion and residues in animal products
The excretion of AfB1 and its metabolites is mainly made through bile liquid and urine. In lactating animals, AfM1 and other metabolites are excreted in the milk. Many studies exist showing the carry-over of aflatoxin into animal products such as eggs, milk and milk products.
In humans, hepatocellular carcinoma (HCC) is a major health problem in China where each year approximately 110,000 patients are diagnosed with it. The HCC cases in China account for almost 45% of HCC incidences worldwide. The mortality rate for HCC is more than 95%. Excluding other risk factors, the consumption of AfB1-contaminated food such as corn, soya-based products, and peanut oil was correlated (r = 0.55) to the HCC fatality rates in people living in ten Chinese villages that were studied. See Yu (1995) for a current review of HCC in China. The high positive correlation between aflatoxin and HCC lead IARC to classify AfB1 as a class 1 human carcinogen.
In 2004, an outbreak of aflatoxicosis in Kenya resulted in 125 deaths from the 317 cases of poisoning. The cause for this 39% fatality rate was contaminated maize with aflatoxin levels up to 8mg/kg. Besides carcinogenic effects, immunomodulatory effects are also observed in the humans along with infectious disease and growth problems in children.
In animals, the effects of aflatoxins are variable depending on sex, age, species and even animal breed. The main target organ for aflatoxins is the liver. Nevertheless, due to the toxins’ interference and reactions with nucleic acids, RNA and DNA, proteins and enzymes, their effects on domestic animals are not only hepatotoxic and expressed by toxic hepatitis and jaundice but involve a broad range of organs, tissues and systems.
Mainly affected systems by aflatoxins
Trichothecenes are a family of over 170 structurally related compounds produced by several Fusarium species. Trichothecenes are mainly classified into two groups:
- Type A (T-2 toxin, HT-2 toxin, diacetoxyscirpenol): characterized by a functional group other than a ketone at C-8
- Type B (Deoxynivalenol, Nivalenol, Fusarenon X): characterized by a carbonyl function at C-8
Among this group deoxynivalenol is the most frequent mycotoxin. The trichothecenes structure is characterized by a sesquiterpene ring and a C -12,13-epoxide ring. T-2 toxin and diacetoxyscirpenol are soluble in non-polar solvents deoxynivalenol or nivalenol in polar solvents like alcohol.
Structural formula of type A trichothecenes
Examples of type A trichothecenes
Structural formula of type B trichothecenes
Examples of type B trichothecenes
Generally, there are three main metabolic pathways:
The de-epoxidation is the most important step in the detoxification of trichothecenes and can be carried out by microorganisms in the gastrointestinal tract of ruminants. Animals which lack this microflora are more sensitive to trichothecenes.
Mechanism of action
Trichothecenes are potent inhibitors of protein, RNA and DNA synthesis and they can interact with the cell membrane. Trichothecenes bind to active polysomes and ribosomes, peptide linkages are interrupted, initiation and termination sequences are reduced, and the ribosomal cycle is disrupted. Their toxicity is primarily based on the 12,13-epoxytricothecene ring. There are two types of the mechanism of protein inhibition:
- Inhibition of the initial step of protein synthesis (e.g.: T-2, HT-2, DAS)
- Inhibition of the elongation-termination step (e.g.: DON)
As potent inhibitors of protein and DNA and RNA synthesis, trichothecenes are especially toxic to tissues with a high cell division rate. Additionally, they are very cytotoxic to eukaryotic cells, causing cell lysis and inhibition of mitosis.
Mechanism of action of deoxynivalenol
Deoxynivalenol enters the cell and binds to active ribosomes which transduce a signal to RNA-activated protein kinase (PKR) and hemoitopoeitic cell kinase Hck. Subsequent phosphorylation of mitogen-actived protein kinases drives transcription factor (TF) activation apoptosis and resultant chronic and immunotoxic effects (modified from Pestka, 2007).
In general, absorption of deoxynivalenol occurs very rapidly within the digestive tract and is widely distributed in many tissues and organs. Currently, there is no evidence for accumulation in tissues or transmission to milk or eggs.
Many outbreaks of acute human disease like vomiting, gastrointestinal disorders, diarrhoea or headaches have been attributed to consumption of Fusarium-contaminated grains. In animals, there are two characteristic effects of deoxynivalenol contamination:
- Decrease in feed consumption (anorexia)
The primary target of T-2 toxin is the immune system and this results for example in changes in the leukocyte count or reduced antibody formation.
Mainly affected systems by trichothecenes
Ochratoxins are metabolites of Aspergillus ochraceus and Penicillium verrucosum in temperate regions and are present in a large variety of feeds and foods. There are four ochratoxins (A, B, C, and D) and the major mycotoxin among this group is ochratoxin A (OTA). This toxin is a contaminant of cereals, beans and other plant products. The most significant effect of ochratoxins in farm animals is nephrotoxicity. Chemically, ochratoxins contain an isocoumarin moiety linked by a peptide bond to phenylalanine.
Mechanism of action
Ochratoxins primarily affect the enzymes involved in phenylalanine metabolism. They inhibit the enzyme involved in the synthesis of the phenylalanine-tRNA complex. Ochratoxins might also interact with other enzymes that use phenylalanine as a substrate. For example the phenylalanine hydroxylase which catalyzes the irreversible hydroxylation of phenylalanine to tyrosine.
In addition, it alters the mitochondrial membrane transportation system and inhibits ATP production and enhanced membrane lipid peroxidation and superoxide and hydrogen peroxide radical formation.
The major metabolite of ochratoxin is ochratoxin A, a hydrolysis product by the gut microflora without the phenylalanine moiety. The other metabolites, the hydroxylated derivates are 4(R)-, 4(S)- and 10-OH-Ochratoxins.
Metabolism of OTA
modified from Pfohl-Leszkowicz et al., 2007
Between 40 and 66% of ochratoxin is absorbed from the gastrointestinal tract depending on the different species. The small intestine has been shown to be the major site of absorption and maximal absorption occurrs in the jejunum.
Ochratoxins bind rapidly to serum albumin and are distributed in the blood mainly in bound form. Generally, the toxin has a long biological half life due to its high rate of binding to serum protein but there are differences between species. Therefore the ochratoxin protein adduct in serum can be used as an biomarker for ochratoxins exposure.
It primarily accumulates in the kidneys followed by the liver, muscle and fat. Due to this accumulation there has been a concern over the potential carryover into meat.
In humans, ochratoxins are suspected of being the main agent responsible for the fatal kidney disease, Balkan endemic nephropathy (BEN) affecting rural populations in the Central Balkan regions, such as Bulgaria or Croatia. This disease is characterized by a decrease in kidney size or tubular degenerations. There is also an association between Balkan endemic nephropathy and urinary tract tumors. According to IARC (International Agency for Research on Cancer) ochratoxins are classified as possible human carcinogens.
In animals, toxicity varies widely according to animal species and sex. The kidneys are the main target organs. Ochratoxins has been found to be responsible for porcine nephropathy, an extensively studied disease.
As well as the above mentioned effects ochratoxin can also have effects on the following systems:
Mainly affected systems by ochratoxins
Fumonisins are a group of recently discovered mycotoxins mainly produced by Fusarium verticillioides and F. proliferatum. They were first isolated from cultures of Fusarium verticillioides in 1988 in South Africa. The most predominant member among this group is Fumonisin B1 (FB1). They mainly occur in maize. Fumonisins are highly polar compounds and soluble in water.
Mechanism of action
Fumonisins toxicity are based on a structural similarity to the sphingoid bases, sphingosine and sphinganine (see figure 1). They are inhibitors of sphinganine (sphingosine) N-acyltransferase (ceramide synthase), a key enzyme in the lipid metabolism, resulting in a disruption of this pathway. This enzyme catalyzes the acylation of sphinganine in the biosynthesis of sphingolipids and also deacylation of dietary sphingosin and the sphingosine that is released by the degradation of complex sphingolipids (ceramid, sphingomyelin and glycosphingolipide).
Sphingolipids are important for the membrane and lipoprotein structure and also for cell regulations and communication (second messenger for growth factors). Sphingosine is the backbone of sphingolipids
As a consequence of this disruption many bioactive intermediates are elevated, others reduced. The main points are:
- Rapid increase of sphinganine (sometimes sphingosine)
- Increase of sphinganine degradation products like sphinganine 1-phosphate
- Decrease of complex sphingolipids
Free sphingoid bases are toxic to most cells by affecting cell proliferation and inducing apoptosis or necrotic cell death or increasing in kidney and serum sphinganine 1-phosphate. The accumulation of sphinganine is associated with hepato- and nephrotoxic effects. Complex sphingolipids are important for cell growth regulation and also cell-cell interactions. Fumonisin B1 impairs the barrier function of endothelial cells in vitro. These adverse effects on endothelial cells could indirectly contribute to the neurotoxicity and pulmonary edema caused by fumonisins. Sphingosine 1-phosphate activates the endoplasmic reticulum calcium release and also acts as a ligand for extracellular receptors in the vasculature (S1P receptors).
The accumulation of free sphingoid bases in the serum and urine is a useful biomarker for the exposure of fumonisins.
Structures of sphinganine, sphingosine und fumonisin B1
Lipid metabolism and the inhibition of fumonisins. In order to simplify the graphics, only the main intermediates are depicted.
modified from Merrill et al., 2001
Exposure and absorption into the organism
Several studies have indicated that fumonisins are poorly absorbed from the gastrointestinal tract and rapidly cleared from the blood. Fumonisins only accumulate insignificantly in the tissues. The most important target organs are the liver and the kidneys depending on species and dosage. It causes apoptosis followed by mitosis in the affected tissues.
Excretion and residues in animal products
There is no evidence for the carryover of fumonisins to eggs. Additionally the exposure of the toxin through milk does not pose a production or health concern to consumers or animals because there is only a minimal carryover.
In humans, fumonisins have been found to cause oesophageal cancer in certain regions (South Africa, China, Italy…) after ingestion of contaminated grains.
According to IARC (International Agency for Research on Cancer) fumonisins are classified as possible human carcinogens.
In the case of animals, horses are the most sensitive species to fumonisin toxicity. The mycotoxin causes a disease syndrome which is called equine leukoencephalomalacia (ELEM) and affects the central nervous system. Several studies have indicated that fumonisins can cause porcine pulmonary edema (PPE). The first sign of fumonisin contamination is often a decreased feed intake. Within 4 – 7 days of being fed contaminated feed pigs show respiratory disorders followed by death due to acute pulmonary edema.
In general, the most affected organs are the liver and kidneys but fumonisins also cause a broad range of effects on other systems.
Mainly affected systems by fumonisins
Zearalenone is an important mycotoxin occurring in warm and temperate climate regions. It is produced mainly by Fusarium graminearum and Fusarium culmorum on a variety of cereal crops.
Metabolism/Mechanism of Action
Zearalenone is an estrogenic mycotoxin which is often involved in reproductive disorders and hyperestrogenicity in farm animals. The estrogenic effects are based on the structural similarity between zearalenone and estradiol. Estradiol is the most important female sex hormone in the group of estrogens.
The biotransformation of zearalenone takes place in two major pathways:
- Hydroxylation: Formation of a-zearalenone and ß-zearalenone, assumed to be catalyzed by 3a- and 3b-hydroxysteroid dehydrogenases (HSDs)
- Conjugation of zearalenone and its metabolites with glucoronic acid catalyzed by uridine diphosphate glucuronyl transferases (UDPGT)
The reduced form of zearalenone, a –zearalenol, has increased estrogenic effects. Several studies have indicated that there are differences in biotransformation of zearalenone in various species. For example pigs convert zearalenone predominately into a –zearalenol, while in cattle there is ß-zearalenol is the dominant metabolite.
Chemical structures of ZEA and its derivatives: (a) zearalenone (ZEA), (b) a –zearalenol (α-ZEA), (c) ß –zearalenone (β-ZEA), (d) zearalanone (ZAN), (e) α-zearalanol (α-ZAL) and (f) β-zearalanol (β-ZAL).
Simplified view of the mode of action of zearalenone (Z)
The mycotoxin passes the cell membrane and binds to the oestrogen receptor. This complex is transferred into the nucleus and binds there to specific nuclear receptors. Afterwards it generates oestrogenic responses via gene activation resulting in the production of mRNAs that code for proteins which are normally expressed by receptor-oestrogen complex binding (modified from Riley and Norred, 1996).
Exposure and absorption into the organism
Zearalenone is rapidly absorbed and metabolized in intestinal cells. In some species zearalenone and its metabolites are detected in the bile.
Zearalenone posses a relatively low acute toxicity and the oral LD 50 values are between 2000–20000 mg/kg b.w.. Effects differ between different species. Pigs and sheep seem to be more sensitive to the effect of zearalenone than others. Furthermore the toxin has shown haematotoxic effects e.g. changes in some blood parameters.
Mainly affected systems by zearalenone
Merrill Jr., A.H., Sullards, M.C., Wang, E., Voss, K.A., and Riley, R.T. (2001) Sphingolipid metabolism: Roles in signal transduction and disruption by fumonisins. Environmental Health Perspectives 109(2), 283-289
Pestka, J.J. (2007) Deoxynivalenol: Toxicity, mechanisms and animal health risks. Animal Feed Science and Technology 137, 283-298
Pfohl-Leszkowicz, A. and Manderville, R.A. (2007) Ochratoxin A: An overview on toxicity and carcinogenicity in animals and humans. Molecular Nutrition & Food Research 51, 61-69
Riley, R.T. and Norred, W.P. (1996) Mechanistic toxicology of mycotoxins. In: The Mycota VI. Human and Animal Relationships (D.H. Howard and J.D. Miller, eds). Springer-Verlag, Berlin, pp.193-211
Yiannikouris, A. and Jouany, J.P., (2002) Les mycotoxines dans les aliments des ruminants, leur devenir et leurs effets chez l'animal. INRA Prod. Anim., 15, 3-16