Clinical and patho-anatomical effects of mycotoxins in animals by agriNews

CLINICAL and PATHO-ANATOMICAL EFFECTS of MYCOTOXINS in animals

R. K. Asrani and Rakesh Kumar Department of Veterinary Pathology, Dr G C Negi College of Veterinary and Animal Sciences, CSK Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India

Mycotoxins are secondary harmful mold metabolites that produce significant detrimental health effects in human beings and animals1. These are low molecular weight compounds known to be harmful even at low concentrations2.

Approximately 25% of the crops, including cereal grains and nuts, are often presumed to be contaminated with fungus3.

The most frequently encountered harmful mycotoxins in foodstuffs and feed include aflatoxin B1 (AFB1), ochratoxin A (OTA), trichothecenes, HT-2 and T-2 toxins, fumonisin B1 (FB1), citrinin (CTN), zearalenone (ZEN) and ergot alkaloids. A predominately marked distribution of fumonisins, zearalenone and deoxynivalenol (DON) is documented globally .

Moisture content (20-25%)

Cereal crops may become contaminated in

Environmental temperature (22-30ยบC) Composition of food items

the field or during harvesting, transport, processing or storage5, 6. The rate of contamination of crops with fungus is more frequently triggered by the rainy season7.

Relative air humidity (70-90%) Physical damage to cereals by pests pH

Factors facilitating the production of mycotoxins in contaminated products include8:

Presence of mold spores

Diseases in animals

BIOLOGICAL FACTORS

Aflatoxixosis (AFB1): liver damage, liver cancer

Susceptible crop

Zearalenone (ZEN): reproductive problems etc.

ENVIRONMENTAL FACTORS Humidity Temperature Moisture Physical damage

Promote fungal growth in crops/ silage/concentrate/ grass, etc.

MYCOTOXINS PRODUCTION

Contaminated feed/fodder intake by animals

Secretion in milk and milk products (AFM1), eggs, meat and meat products (sausages)

HARVESTING, STORAGE & PROCESSING Consumption by humans (Mycotoxicosis)

Improper moisture, maturity and temperature conditions

Figure 1. Mycotoxins production and their occurrence in the food chain.

Common routes of entry of

Mycotoxins are known to produce several

mycotoxins into the body are:

harmful effects in animals and human beings. Classification of these toxins can be made on

Direct consumption of contaminated

the basis of toxicity13 and clinical symptoms

products of plant origin (cereals, nuts,

related to the organs damaged14.

bread etc.) and products obtained from animals (meat and meat products, milk,

Highly toxic (lethal at 1-10 mg/Kg body weight) Trichothecenes Aflatoxin B1 Citreoviridin

offalâ&#x20AC;&#x2122;s, fermented sausages etc.)9, 10. Dermal contact and inhalation are not very common routes but can act as a potential mode of entry into the body11. Harmful toxic effects of mycotoxins depend on11,12:

Severely toxic (fatal even < 1 mg/ Kg body weight) Rubratoxin B Cyclochilorotine

MYCOTOXINS

Type of mycotoxin Dose introduced into the body Duration of exposure to the mycotoxins

Figure 2. Classification of mycotoxins on the basis of toxicity.

All other Mycotoxins (toxic > 10 mg/Kg body weight)

Heapatotoxicity (aflatoxins, rubratoxins, fumonisins, cyclochlorotine)

Nephrotoxicity (ochratoxins, citrinin, quinones, viomelein, xanthomegnin)

Figure 3. Classification of mycotoxins on the basis of clinical manifestations. Neurotoxicity (fumonisin, patulin, citreoviridin)

MYCOTOXINS Estrogenicity (zearalenone)

Photosensitivity (sporedesmins)

Cytotoxicity (trichothecenes)

Immunotoxicity (ochratoxins, trichothecenes)

Table 1. Summary of different Mycotoxins with their toxic effects

Name of the fungus

Mycotoxins

Acronyms

Common sources

Target organs

Pathological change/Disease condition

Species susceptibility

Mode of action

References

Aspergillus ochraceus, A. verrucosum, Penicillium nordicum Aspergillus flavus, A. parasiticus

Ochratoxin

OTA

Kidneys, liver

Renal damage and cancer, hepatotoxicity

Swine, poultry, quail, human beings

Protein synthesis inhibition, nucleic acid damage and lipid peroxidation

15, 16, 17

Aflatoxins

AFB1, AFG1, AFB2, AFG2, AFM1

Liver, gastrointestinal system

Liver damage, hepatocellular carcinoma (HCC)

Pigs, human, dogs, cats, poultry

DNA adducts formation, mutations, inhibition of protein synthesis

18, 19

Trichothecium spp., Stachybotrys sp., Fusarium sp.

Trichothecenes

T-2, DON

Coffee, cereal grains, peanuts, dried fruits, cocoa, wine, spices, grape juice Sorghum, soybeans, nuts, rice, corn, cotton seed, cocoa beans, barley, dried fruits, crude vegetable oil Rye, wheat, barley, millet, oats

Gastrointestinal system and skin

Skin and gastrointestinal disorders

Horse, pigs, poultry, cattle, human beings

20, 21, 22, 23, 24, 25

Fusarium verticillioides, F. avenaceum, F. Tricinctum Fusarium graminearum, F culmorum Fusarium cerealis, F. roseum, F. incarnatum Fusarium proliferatum, F. verticillioides

Moniliformin

MON

Wheat,maize, rice,oats

Heart

Heart problems, depression

Birds

Inhibition of mitochondrial translation, inhibition of protein synthesis, DNA fragmentation Inhibition of thiamine pyrophosphatase pyruvate dehydrogenase

Zearalenone

ZEN

Wheat and maize bran

Reproductive system

Abortions,estrus defects, malformation of genital organs

Pigs, bovines, human beings

Binding with estrogen receptors, blocking the secretion of steroid hormones and suppressing estrogenic responses

Fumonisins

FB1, FB2

Silage, corn, wheat, barley, rice

Brain, lungs, esophagus

Swine, human beings, horses

Ergocristine, ergotamine, ergocryptine, ergometrine

Ergot alkaloids

Rye, wheat, triticale, barley, millet, oats

Smooth muscle, nervous system

Inhibition of sphingolipid biosynthesis, inhibition of protein synthesis in eukaryotic cells Partial agonist and weak antagonist in smooth muscles of the body, including blood vessels, and antagonist in central nervous system

Claviceps purpurea

Pulmonary oedema in pigs, Leucoencephalomalacia in equines Dry gangrene, abortion, hallucinations

Cattle, horse, sheep, poultry, human beings

26, 27

30, 31, 32

Effects of AFLATOXINS exposure Episodes of aflatoxicosis are

The order of severity of the mutagenic, immunosuppressive

associated with the production

and carcinogenic effects of aflatoxins is:

of aflatoxins by common fungal species such as Aspergillus

flavus and A. parasiticus in contaminated food products33.

AFB1> AFG1> AFB2>AFG233 AFB1 is predicted to exhibit developmental defects along with immune system dysfynction38.

In 1960, in the UK, the first report of mortality caused by aflatoxins-contaminated groundnut meal in turkeys

Hepatotoxicity

Teratogenicity

Immunotoxicity

Mutagenicity

Carcinogenicity

AFB1

and poultry was reported34. The list of aflatoxins produced by

Figure 4. Harmful effects of AFB1

several fungal species includes AFB1, AFB2, AFG1, AFG2 and AFM1.

Species susceptibility to aflatoxins

Among all known aflatoxins, AFB1 is the most common and potent35.

All animal species are sensitive to aflatoxicosis, but outbreaks are usually encountered among pigs, cattle and sheep39.

Aflatoxins are very stable and are rarely destroyed after

The significant economic losses, including decline in growth rate

processing36. Additionally,

and productivity, are usually reported in farm animals depending

residues of aflatoxins

on individual susceptibility and the targeted species40, 41, 42.

are also reported to be excreted in milk, milk

Chronic exposure to AFB1 in farm animals can lead to various

products, meat and eggs33.

ailments, including liver dysfunction, compromised immune status and susceptibility to several diseases43,44,45,46,47,48.

AFB1 is well recognized for its hepatotoxic, teratogenic, immunotoxic and mutagenic

Some of the animal species, such as monkeys, chickens and

potential and is classified as group

mice have been found to be resistant to AFB149, whereas cattle,

1 carcinogen by International

horses and sheep are quite prone to AFB1-induced toxicity.

Agency for Cancer Research (IARC)37, as it causes hepatocellular

Younger animals have proven to be more

carcinoma in human beings.

susceptible than adult and older animals50. Among aquatic animals, trout have been observed to be the most sensitive to AFB1 toxicity51. Among poultry, the order of sensitivity is: ducks > turkeys> Japanese quail> chickens

AFB1

Metabolized by cytochrome P450 (CYP) in the liver to AFB1-8,9-exo-epoxide (AFBO), AFM1, aflatoxicol (AFL), AFB2a, AFQ1, AFP153

AFB1-8,9-exo-epoxide (toxic derivative and electrophilic in nature)

Binding with guanine residues of DNA and RNA in hepatocytes

Adducts induce mutations in DNA and inhibit DNA transcription and RNA translation54,55

Figure 5. Flow chart indicating mode of action of aflatoxin B1

Abdominal pain, vomiting and oedema can be observed in acute stages, whereas development of hepatocellular carcinoma is evident in later stages56.

AFB1 toxicity in ruminants leads to: Decline in ruminal motility Decline in the cellular digestion and fatty acid production Decline in feed efficiency and is secreted in milk as AFM1 after 12 h of consumption.

Aflatoxin M1

Aflatoxin M1 (AFM1) is a group 1 carcinogen

(IARC) formed through CYP1A2-dependent hydroxylation microbial biotransformation from AFB1. The nuclear adducts are formed and secreted in milk and urine.

Aflatoxin B1 contaminated feed ingested by animals

Microbial biotransformation (CYP1A2-dependent hydroxylation) to AFM1

DNA adducts

Excretion in milk (DAIRY PRODUCTS) and urine Conjugation with glucuronic acid and excreted in bile

The concentration of AFM1 in milk is influenced by several factors, such as duration of lactation and the milk yield of the animal57.

Table 2. Permissible limits of aflatoxins consumption58, 59. Agency

Maximum permissible limit of aflatoxins

AFM1 in milk as per US Food and Drug Administration (FDA)

0.5 μg/kg

Maximum permissible limit of AFM1 in milk and dairy products as per European Commission (EC)

50 ng/kg

Maximum permissible limit of AFB1 in dairy feed (FDA)

20 μg/kg

Health risk to infants and the human population

*An average intake of aflatoxins in human beings ranges between 10-200 ng/kg/day

Figure 6. Aflatoxin M1 in the food chain

Table 3. Clinical and patho-anatomical effects of aflatoxins.

Species

Clinical symptoms

Pathological changes

References

Humans

High fever, vomiting, tremors, hypoglycemia, coma and dark colored urine. Elevated levels of AST, ALT, ALP, creatinine, catalase, malondialdehyde (MDA) and declined values of total proteins, magnesium and reduced glutathione. Depression, anorexia, fever and ruminal contractions in a study conducted by Elgioushy et al.61. Acute aflatoxicosis in Hereford calves after the consumption of peanut hay containing 2230 µg AFB1/kg led to symptoms including icterus, photosensitization, diarrhoea, depression and anorexia by a study conducted by McKenzie et al.62. More sensitive to aflatoxicosis Contaminated feed consumption leads to reduced feed intake, declined growth and production status64.

Cerebral oedema, hemorrhages, fatty degeneration in liver and kidneys, encephalopathy, cirrhosis and hepatocellular carcinoma (HCC). Liver enlargement, distended gall bladder, congested intestine with congestion of the kidneys61. Proliferation of connective tissue involving portal triads in chronic toxicity of feed contaminated with aflatoxins63.

33, 60

Pregnant sows: distorted hepatic architecture, hemorrhages, distended sinusoids, cystic spaces in the liver, lymphoid depletion in lymph nodes and spleen65. Hyperacute cases: hepatic necrosis and hemorrhages. Acute toxicity cases: cellular infiltration, swollen hepatocytes with cholestasis. Subacute toxicity cases: vacuolar degeneration, cholestasis with profound bile duct hyperplasia66. Hydropic degeneration in the hepatocytes, necrotic changes, hyperemia, sinusoidal contraction with accumulation of ceroid pigments have been observed in macrophages from Merino rams treated with aflatoxins at the dose of 250 µg/day68. Hemorrhagic spots in muscles, atrophied spleen; enlarged, paler, fatty liver with hemorrhagic areas; distended gall bladder, nephropathy and thickened crop and proventricular mucosa. Microscopic fatty changes in hepatocytes, acinar arrangement of hepatocytes, lymphocytes and heterophilic aggregates with multiple areas of necrosis, hyperplastic changes in crop and proventricular mucosa70,71.

64, 65, 66

Cattle

Swine

Sheep

Decreased erythrocyte and leucocyte count with decreased values of hemoglobin and packed cell volume before initiation of clinical manifestations67.

Poultry

Feeding aflatoxins at a rate of 3.5 mg/kg of feed resulted in a marked decrease in body weight and growth performance with increased kidneys and liver weight69.

61, 62, 63

67, 68

69, 70, 71

Image 1. Gross pathological alterations associated with AFB1. Liver of a rabbit showing chronic hepatitis along with tumorous growth.

Image 2. Photomicrographs of pathological alterations associated with AFB1

a. Liver showing diffuse hemorrhages along with necrotic area in the hepatocytes along with hemosiderin deposition (H&E*66). b. Liver showing swollen hepatocytes with hydropic changes (H&E*66). c. Photomicrograph of liver showing portal fibrosis with bile duct hyperplasia (H&E*33). d. Liver showing peripheral shifting of nucleus giving a signet ring appearance indicating fatty changes in hepatocytes (H&E*66).

Effects of OCHRATOXIN A exposure Aspergillus ochraceus, Auplopus

This mycotoxin was first reported

Pigs and poultry more

carbonarius and Penicillium

in contaminated cornmeal

sensitive to OTA

verrucosum are the most common

it is considered to be the most

fungal species associated with

common and potent mycotoxin

the production of ochratoxins

produced by these fungi73.

and

induced toxicity. Ruminants are usually resistant, as OTA is degraded

in contaminated grains, raw and cooked food items and beverages

OTA is readily known for its

by ruminal microflora to

(coffee, beans, and wine).

nephrotoxic, carcinogenic,

less toxic metabolites

immunosuppressive, teratogenic

such as OTAÎą78.

Aspergillus ochraceus and

and genotoxicity in animals74,75,76.

Penicillium verrucosum are the

Additionally it has been found

Some researchers have shown

most potent moulds responsible

to produce hepatocellular

the release of OTA in breast

for the production of Ochratoxin

carcinoma as well, apart from

milk, which means it can act as

A (OTA) in tropical and

the nephrotoxic properties, in

a potent threat to the newborns

temperate regions, respectively.

a dose dependent manner .

through breastfeeding79.

Intake of OTA contaminated food items

Binding of OTA to blood albumin

Proximal convoluted tubule (PCT) (Target site)

Organic anion transporter (OAT) 1 and 3 help in the absorption of OTA in the interstitium and OAT 4 in the tubular lumen

Production of reactive metabolites that form adducts after reacting with DNA Alteration of the transmembrane potential of the mitochondrial membrane, causing the release of cytochrome c and apoptosis Inhibition of protein synthesis by competing with Phe-tRNA synthase

Figure 7. Flow chart indicating the mechanism of action of OTA in kidney tubular cells80 81 82.

Table 4. Tolerable limits of OTA. Agencies

Tolerable limits of OTA

Joint Expert Committee on Food Additives (JECFA)84

112 ng/kg body weight/week

Maximum limit of OTA (As per EU)85

3 Îźg/kg in processed cereal

European Food Safety Authority83

17 ng/kg body weight/day

*In one of the studies in Italy from Capei et al.83 has documented an 8% contamination of OTA in breakfast cereals and 50% contamination in sweet snacks with a contamination limit ranging between 2.9 â&#x20AC;&#x201C; 8.6%. A daily intake of OTA at the dose of 1 mg/Kg body weight for 5-6 days can be harmful.

Table 5. Clinical and patho-anatomicaleffects of ochratoxins.

Species

Clinical symptoms

Pathological changes

Referencias

Humans

Weakness, brown discoloration of the skin and lumbar pain. Biochemical parameters such as glucose, gamma-glutamyl transferase and leucine aminopeptidase increase in urine, along with proteinuria with RBCS and WBCs in urine86.

86, 87, 88, 89, 90, 91

Swine

Bulgarian and Danish porcine nephropathy, reduced feed intake and weight gain92. Residues can be transported to human beings through pork or offals93.

Poultry, quail and rats

In poultry, they cause a decrease in egg production, decreased FCR, immunosuppression, developmental abnormalities, reduced feed consumption with an increased water intake49.

Acute renal failure , Balkan endemic nephropathy (BEN), Tunisian nephropathy88. BEN is a tubule-interstitial renal disease that leads to contracted kidneys in later stages86, which may also be followed by renal tumours89. Tubular and glomerular degenerations with fibrotic changes in interstitial tissue, necrosis, apoptosis and end stage kidney damage90,91. Swollen, pale, firm kidneys with gross lesions of fibrosis Microscopically, degenerative changes with fibrosis in the kidneys of pigs from abattoirs during random sampling were consistent finding in post weaning multisystemic wasting syndrome (PMWS), nephropathy syndrome (PDNS) and ochratoxicosis94. Diffuse tubular nephrosis and interstitial fibrosis95. OTA is found to majorly affect mitochondria in PCT cells and also causes degranulation of the rough endoplasmic reticulum (RER). The changes associated with damage of kidneys in quail include profound karyomegaly, cellular swelling, cytoplasmic vacoulations, margination of chromatin material96. Changes such as bile duct hyperplasia, necrosis of liver cells, vacuolar degenerations, dilation of central veins and sinusoids, along with mononuclear cell (MNCs) infiltration has also been reported in the liver by a research concluded by Patial et al.97. Abnormalities of the central nervous system (CNS) along with skeletal system defects were reported in rats when administered OTA during the gestation period98. 87

92, 93, 94, 95

49, 96, 97, 98

Image 3. Gross pathological alterations associated with OTA. a. Swollen and pale kidneys of Japanese quail (right) after administration of Ochratoxin A in diet in comparison to the kidneys on the left side b. Ruffled appearance of feathers in a Japanese quail after feeding Ochratoxin A.

Image 4. Photomicrograph of pathological alterations associated with OTA. Kidney showing fibrous tissue accumulation in the interstitial tissue causing atrophy of renal tubules in OTA toxicity (H&E*66).

Effects of FUMONISIN exposure Fumonisins are produced

Toxicity associated to fumonisins

Fumonisins are also reported

by fungal species such as

was firstly reported in 1980 as a

to cause leukoencephlomacia

Fusarium verticillioides and F. proliferatum, and they are frequently spotted on maize giving it a whitish appearance99.

cause of equine encephalomalacia

in horses, hepatocellular

(ELEM) and porcine pulmonary

carcinoma in rats and pulmonary

oedema (PPE) in the United States,

oedema in association with

and esophageal cancer in Africa.

hydrothorax in pigs103, whereas the IARC has also documented

The most common forms of

These mycotoxins cause

the carcinogenic potential of

fumonisins include fumonisin A and

neurotoxicity, hepatotoxicity,

fumonisins in human beings104.

fumonisin B (B1, B2, B3 and hydrolyzed

embryo toxicity and

B1), and among these fumonisin B1

nephrotoxicity in animals101,102.

is the most common and potent

100

As per JECFA, the maximum tolerable limit of FB on the basis of no-observable-effectlevel (NOEL) of 0.2 mg/kg The production of this

bw/day with a safety factor

mycotoxin is promoted when

100 is 2 Îźg/kg/day105.

moisture content is < 19%.

Mechanisms of action of Fumonisins102: Competitive inhibition of the ceramide synthase enzyme Oxidative stress and endoplasmic reticulum stress

Figure 8. Mechanisms of inhibition of sphingolipid metabolism.

Autophagy modulation Alteration of DNA methylation

Competitive inhibition of the ceramide synthase enzyme Required for sphingolipid biosynthesis

Disruption of sphingolipid metabolism

In liver, kidney and brain tissue

Inhibition of the acylation of sphingosine and sphinguanine

Inhibition of ceramide synthase

Table 6. Clinical and patho-anatomical effects of fumonisins. Species

Clinical symptoms

Pathological changes

Reference

Humans

Esophageal cancer106.

106, 107

Cattle

Optic nerve degeneration leading to blindness108.

Swine - Porcine pulmonary oedema (PPE)

Defective vision, staggering, drowsiness, weight loss, decreased feed intake, respiratory distress and cynosis110,111,112.

Horses - Equine leukoencephalomalacia (ELEM)/Moldy corn poisoning) Fish

Circling, head pressing, blindness, ataxia and depression110.

Liquefactive necrosis or softening of cerebral subcortical white matter with occasional areas of hemorrhages. Histological examination shows eosinophilic and swollen astrocytes in white matter of brain. Pregnant female mice treated with 2.5 or 10 mg/kg FB1 intraperitoneal injection show neural tube defects in fetuses107. Liver and kidney damage have been reported in calves treated with 1 mg FB1/kg body weight for 7 days109. Histological changes evidenced in cattle with blindness includes fibrosed septa, retinal degeneration, optic nerve degeneration and oedema of myelin108. Grossly, it is characterized by the presence of fluid in the thoracic cavity and airways with widened interlobular septa. Microscopic evaluation depicts widened interlobular septa with perivascular and peribronchiolar oedema along with MNCs infiltration. The right ventricle of the heart and pulmonary artery shows hypertrophy. Vacuolar changes and cellular swelling of hepatocytes is also evident111, 113. Focal to multifocal areas of necrosis of white matter. Degenerative changes in endothelial cells along with perivascular thrombosis. Oedema formation in neural tissue with neutrophilic infiltrations114.

Birds

Decrease in body weight, increased serum biochemical markers in ducks116. Diarrhoea, increased gizzard, proventriculus and liver weight in broilers117.

Nervous symptoms115.

Chronic exposure of one year old carp (Cyprinuscarpio. L) with a feed containing FB1 at the dose of 10 mg/kg body weight for 42 days showed nervous manifestations.Histological evaluation of brain tissue reflected degenerative changes, vacoulations, necrotic changes in the brain cells around periventricular area and capillaries115. Liver of affected quail showed necrotic hepatitis along with infiltration of heterophils and macrophages. Increased Kupffer cell activity, bile duct hyperplasia with increased granularity of cellular cytoplasm has also been evidenced118. Additionally, kidneys showed swelling of the tubular epithelial cells and glomerular tuft, obliteration of Bowmanâ&#x20AC;&#x2122;s capsule, apoptotic changes and elevated mitotic activity118. Deshmukh et al.119 concluded progressive degenerative changes along with heterophilic infiltration in tubular epithelial cells and interstitial tissue of kidneys at the dose of 150 ppm for 21 days in quail. Hepatic necrosis, bile duct hyperplasia, thymic atrophy and rickets in broiler chicks affected with FB1 has also been observed in a study conducted by Ledoux et al.117.

108, 109

110, 111, 112, 113

110, 114

115

116, 117, 118, 119

Image 5. Gross pathological alterations associated with FB1. a. Enlarged liver of a Japanese quail after feeding Fumonisin (FB1) for 3 weeks at the dose of 300 ppm. b. Enlargement of liver (right side) with Fumonisin (FB1) toxicity in comparison to normal liver on the left side.

Image 6. Photomicrograph of pathological alterations associated with FB1. Liver of a Japanese quail reflecting necrotic changes along with heterophilic infiltration admixed with mononuclear cells after the administration of FB1 (H&E*330).

Effects of TRICHOTHECENES exposure Trichothecenes are toxic secondary

The main mycotoxins belonging to

metabolites produced by Fusarium

the trichothecene group include type

graminearum, Stachybotrys, Fusarium poae, Fusarium langsethiae, etc, often found contaminating wheat, maize, barley and oat kept in damp environmental conditions.

A (T-2) and type B toxins (DON), and their toxic potential is due to the presence of an epoxide ring122. These toxic metabolites are quite resistant to processing and are only destroyed at temperatures above

Production of these

260ยบC for more than 30 min.

mycotoxins is often favored by ambient temperature (0-32oC) with humid conditions120, 121.

Harmful effects and tolerable limits of trichothecenes

In pigs, cattle, broilers and rats, trichothecenes

According to the EU, the maximum limit for the

damage the liver and stomach123.

presence of DON in cattle feed is 5 mg/Kg feed, whereas it is around 1 mg/Kg feed for calves.

Therefore, trichothecenes toxicity in farm animals is often associated to symptoms such as vomiting,

In studies conducted by Ingalls129 and Cote et al.130

diarrhoea, anorexia, weight loss and death

no marked variation in the milk production was

124,125

Additionally, the malabsorption induced by

reported when DON is given at a rate of 14 mg/kg

trichothecenes in pigs, poultry and rats is often

for 3 weeks and 66 mg/kg for 5 days, respectively.

associated with necrosis of intestinal villi126,127. Based on the presence of ester-ether bonds between C-4 and C-15 at C-12 we can divide trichothecenes into 2 types: macrocyclic and non-macrocyclic. The nonThe maximum tolerable limits of

macrocyclic trichothecenes are enlisted in Table 7.

DON in most parts of the world are limited to 0.75 mg/kg in human diets and 1-5 mg/kg in animal rations128.

Table 7. Classification of trichothecenes.

A* Non-macrocyclic trichothecenes B

T2 toxin Diacetoxyscirpenol (DAS) Neosolaniol Nivalenol Deoxynivalenol (DON) Fusarenon-X

*T2 and DAS are used as bioweapons and are mainly produced by Fusarium poae and Fusarium langsethiae.

Inhibition of protein synthesis after binding to the 60S subunit of ribosome, leading to the inhibition of peptidyltransferase and inhibition of the initiation, elongation or termination steps in protein synthesis

Oxidative stress mediated DNA damage and apoptosis

TRICHOTHECENES

Figure 9. Clinical and patho-anatomical effects of trichothecenes20, 21, 22, 23.

Inhibition of mitochondrial translation

Table 8. Clinical and patho-anatomical effects of trichothecenes. Species

Clinical symptoms

Pathological changes

References

Humans

Type A trichothecenes can lead to alimentary toxic aleukia (ATA) in human beings causing severe leukopenia, vomiting and nervous symptoms131,132. Production losses, altered reproductive potential, decline in liver function and immunosuppression is seen in dairy cattle fed with silage and cereal grains contaminated with trichothecenes133. Calves affected with DON toxicity show icterus with altered liver enzymes134. Growth performance and feed intake in finishing pigs is severely influenced by DON toxicity138.

Skin rashes, necrotic stomatitis, hemorrhagic vaginitis and nervous system affections131.

131, 132

Postmortem evaluation of dead carcasses reveals congestion and hemorrhages in abomasum, splenomegaly and kidney damage. Histological investigation often reflects degenerative changes, cholestasis, bile duct hyperplasia, steatosis and infiltration of mononuclear cells (MNCs) especially macrophages and lymphocytes134. T-2 toxicity in cattle leads to absence of estrus, hemorrhagic and necrotic enteritis, decreased feed intake and reduced milk production135,136,137.

133, 134, 135, 136, 137

Piglets fed with DON (1.5-2.8 mg/kg feed for 4-5 weeks) were found to show gastrointestinal and hepatotoxic pathological alterations139, 140. DON interferes with reproductive potential of sows as is speculated to produce harmful effects on the ovaries and follicles141. Feed contaminated with T2 toxin in acute toxicity cases lead to myocarditis/cardiotoxicity, rumenitis with ulcerative abomasitis, anasarca, brain oedema and pancreatic necrosis142.

138, 139, 140, 141

Providing T-2 toxin contaminated feed (10-20ppm) in juvenile goats led to severe pathological alterations in the intestine and liver on ultra-structural evaluation. Apoptotic changes are markedly prominent in mesenteric lymph nodes, proximal convoluted tubules (PCTs) and distal convoluted tubules (DCTs) of the kidneys, enterocytes in the intestine and spleen with significant up-regulation of pro-apoptotic proteins, HSPs and cytokines143. The histological changes in the liver tissue of goats included centrilobular necrosis, sinusoidal congestion, peri-ductular connective tissue proliferation, bile duct hyperplasia and vacuolar degeneration143.

143

Cattle

Swine

Sheep

Goats

Chronic exposure is often followed by declined reproductive potential, gastroenteritis, weight loss, myocarditis and pus in the oral cavity142. Growth retardation, lethargy, decrease platelet, Hb and total leucocyte count, decrease in the values of serum and tissue superoxide dismutase and catalase143.

142

Effects of ZEARALENONE exposure The most common fungal species involved in the production of zearalenone (ZEN) include Fusarium

culmorum, F. cerealis and F. graminearum. This mycotoxin is commonly found in cereal grains in temperate regions with warm weather144, 145 and can remain stable at temperatures up to 150˚C146. The highest production of ZEN is reported at 25˚C with 16% moisture content147,148. Five major metabolites of ZEN include α-zearalenone (α-ZEN),

Pigs are speculated to be the most sensitive species for ZEN-induced

β-zearalenone (β-ZEN), α-zearalenol

reproductive disorders as compared to other animals157.

(α-ZAL), β-zearalenol (β-ZAL) and zearalenol (ZON), α-ZEN having the

About 80-85% of oral dose of ZEN is found

highest estrogenic activity

to be efficiently absorbed in pigs158.

149, 150

Zearalenone is responsible for causing ear rot in maize and head blight in wheat and barley151, with immunotoxic, genotoxic, hepatotoxic and hematotoxic effects in animals, as well as significant nephrotoxic potential

The concentration of ZEN and α-ZEN in follicular fluid of swine is 38.9 and 17.6 pg/ml, respectively159. Very limited data is documented about the folliculogenesis in ovaries of domestic animals160, but ZEN shows affinity towards estrogen receptors in uterus, mammary gland, brain and bones, which reflects its estrogenic potential161.

with an ability to produce pituitary adenomas152,153,154,155. Additionally, ZEN is linked to reproductive disorders in animals and hyperestrogenic syndrome in human beings156.

Table 9. Tolerable limits of zearalenone (ZEN). Tolerable daily intake (TDI) in humans162 Cereals151

20-200 μg/kg Processed cereals

75 μg/kg

Unprocessed cereals

100-200 μg/kg

Unprocessed cereal snacks

50 μg/kg

Cereal foods

20 μg/kg

Regulatory limits (China) in wheat/corn/flour163

60 μg/kg

Figure 10. Mechanism of action of ZEN

Oral intake of ZEN

Absorption through the gastrointestinal tract

Converted to active metabolites (α-ZAL, β-ZAL) with the help of 3α and 3β hydroxysteroid hydrogenase

Binding to estrogen receptors in uterus, mammary gland, brain and bones, so reflecting its estrogenic potential

ZEN blocks the secretion of steroid hormones and suppresses the estrogenic response in the preovulatory phase

Table 10. Clinical and patho-anatomical effects of Zearalenone. Species

Clinical symptoms

Pathological changes

References

Humans

Hypoestrogen syndrome in human beings, acting as a stimulating factor for precocious puberty development in females164. Genotoxicity effect on the lymphocytes due to the formation of DNA adducts167,168. ZEN is excreted in the milk of cows fed with high doses of ZEN. Heifers fed with 99% pure ZEN at a rate of 250 mg/day showed a decline in the conception rate of 87-62%169. Weaned gilts affected with ZEN toxicity usually reflect ovarian atrophy, vulvar hypertrophy without any significant effects in uterus and mammary glands170,171. Hormone production and estrus cycle length is not altered in mares provided with oats contaminated with ZEN (2 mg/ Kg)160. ZEN leads to a decline in serum progesterone and testosterone concentration, reduced sperm count, increased incidence of infertility and decreased conception rate in pigs, cows, rats and mice168,174.

Endometrial hyperplasia, mammary tumors, adenocarcinoma and proliferative changes in women165,166.

164, 165, 166

Irregular estrus, infertility, abortion, retention of placenta, mastitis and metritis.

167, 168, 169

Decreased fertility, abnormal estrus cycle, abortion, vulvovaginits and reduced litter size172.

170, 171, 172

Ovarian follicular atresia173.

160, 173

Cystic mammary glands, hepatopathy and nephropathy, uterine fibrosis, persistent estrus, sterility, squamous metaplasia and, hyperplasia of the endometrial glands175,176.

168, 174, 175, 176

Cattle

Pigs Horses Rodents

Effects of MONILIFORMIN exposure Fungal sources involved in the production of monilformin (MON) include Fusarium

moniliforme, F proliferatum, F. avenaceum, F. subglutinans, F. tricinctum and Pencillium melanoconidium177, 178, 179, 180. Contaminated cereal grains and plants used for silage

Inhibition of thiamine pyrophosphatase enzymes in tricarboxylic acid cycle

preparation are the major source of production of this mycotoxin. MON is cardiotoxic and hematotoxic , with acute toxicity 181

Altered oxidation of pyruvate and Îą-ketoglutarate

that is comparable to trichothecene toxicity (T2, HT-2)182, 183. Fatal outbreaks of MON are reported in animals, but experimental studies in birds and rats have shown potential pathological effects184,185,186.

Inhibition of synthesis of collagen type II and aggrecan causing catabolic effect on articular cartilages

Inhibition of pyruvate dehydrogenase

Figure 11. Mechanisms of action of MON187, 188.

Clinical and patho-anatomical effects of moniliformin

In birds and laboratory rodents, intestinal hemorrhages are seen in acute cases, whereas cardiac hemorrhages are typical lesions in sub-acute and chronic cases of MON toxicity189. In one of the sub-acute toxicity studies conducted by Jonsson et

al.190 reflected intestinal hemorrhages with pulmonary congestion in rats without other specific lesions in other organs. Cardiomyopathy depicted by necrotic and degenerative changes in the heart with hypertrophy of muscle fibers causing cardiac arrest in quail birds fed with MON at the dose of 100 ppm has also been documented in previous studies191.

Image 7. Gross pathological alterations associated with MON. Japanese quail showing rounding and dilation of heart (Right side) after feeding MON at the dose of 110- ppm for 3 weeks; Left side showing normal heart.

Image 8. Photomicrographs of pathological alterations associated MON. a. Heart of a Japanese quail showing hypertrophy of cardiac muscle fibers following MON administration (H& E*132). b. Glomerular tufts occupied by needle shaped uric acid crystals in MON toxicity (H&E*66).

MULTI-MYCOTOXIN toxicity

In field conditions, it is most common to find raw materials to be contaminated with one or more mycotoxins, with variations in the symptoms associated with exposure, as the combination of these toxins can involve different types of interactions, such as synergistic, additive or antagonistic effects as shown in Table 11.

Table 11. Combined toxic effects of various mycotoxins. Mycotoxin

Combination and type of interaction

Toxic effects produced

Ochratoxin (OTA)

Citrinin + FB1 (additive and synergistic)

Cytotoxicity to mononuclear cells192

FB1 (additive and synergistic)

Nephrotoxicity, hepatotoxicity, genotoxicity and immunosuppression

ZEN (antagonistic)

Cytotoxicity161, 197

Citrinin (synergistic, antagonistic and additive)

194, 196

Trichothecenes (synergistic and additive)

Nephrotoxicity, immune organ depletion, gastrointestinal problems and fetal malformations194, 196 Nephrotoxicity, immunotoxicity and hepatotoxicity198, 199

AB1 (synergistic and antagonistic)

Nephrotoxicity, teratogenicity, hepatotoxicity and cardiotoxicity

196, 200

MON (Synergistic)

191, 201

ZEN (antagonistic and synergistic)

Cardiotoxicity, nephrotoxicity, hepatotoxicity, immunosuppression, respiratory distress191, 201 Cytotoxicity and immunostimulation202

Trichothecenes (antagonistic and synergistic)

203, 204

Neural tube defects, hepatotoxicity, esophageal cancer

203, 204

Trichothecenes + ZEA (synergistic, antagonistic and additive

Cytotoxicity, oxidative damage and blockade of synthesis of macromolecules204, 205

204, 205

Fumonisin B1 (FB1)

References 192 193, 194,

195, 196

196, 200

193, 194, 195, 196 161, 197

198, 199

202

*The combined harmful effects of different mycotoxins depend upon the absorption rate206

CONCLUSIONS Mycotoxins are very harmful

Although in many of the countries

In order to limit the production

metabolites known to contaminate

tolerable limits for various

of mycotoxins, several strategies

food items and are majorly

mycotoxins are standardized,

are proposed and followed time

implicated in several clinical

a wide range of developing

and again by various agencies and

and pathological impairments in

regions around the globe still

regulatory bodies. In the present

human beings and animals.

need a thorough establishment

scenario, to minimize the production

of such standards with a strict

of mycotoxins during processing of

follow-up to reduce the levels of

raw material and final food products

mycotoxins in the food chain.

for animal or human use the basic principles to be followed include:

It is of utmost concern to prevent fungal contamination of food Excessive levels of mycotoxins can cause health hazards to the animals directly and through animal products to human beings.

products by providing high quality crops or animal products with controlled storage, harvesting and distribution strategies.

Good Agricultural Practices (GAP) Good Manufacturing Practices (GMP) Hazard Analysis Critical Control Points System (HACCP)

Regular monitoring of food items, animal feed etc. by employing proper guidelines and safety standards definitely will help to limit the fungal contamination.

BIBLIOGRAPHY 1. Haschek, W.M.; Voss, K.A. Mycotoxins. Haschek and Rousseaux’s Handbook of Toxicologic Pathology. Third Edition. University of Illinois, Urbana, IL, USA, 2 USDA Agricultural Research Service, Athens, GA, USA, 2013; 1187-1258. http://dx.doi.org/10.1016/B978-0-12-4157590.00039-X 2. Milićević, D.R.; Skrinjar, M.; Baltić, T. Real and perceived risks for mycotoxin contamination in foods and feeds: challenges for food safety control. Toxins 2010, 4, 572-92. doi: 10.3390/toxins2040572. 3. Pandya, J.P.; Arade, P.C. Mycotoxin: a devil of human, animal and crop health. Adv. Life Sci. 2016, 5, 3937–3941. 4. Binder, E.M. Managing the risk of mycotoxins in modern feed production. Animal Feed Science and Technology 2007, 133 (1–2), 149-166. 5. Coffey, R. EndaCummins; ShaneWard. xposure assessment of mycotoxins in dairy milk. Food Control 2009, 20 (3), 239-249. 6. Khazaeli, P.; Najafi, M.L.; Bahaabadi, GA.; Shakeri, F.; Naghibzadeh tahami, A. Evaluation of aflatoxin contamination in raw and roasted nuts in consumed Kerman and effect of roasting, packaging and storage conditions. Life Sci. J. 2014, 10, 578–583. 7. Pleadin, J.; Vahcic, N.; Persi.; Sevelj.; Markov, K.; Frece, J. Fusarium mycotoxins’ occurrence in cereals harvested from Croatian fields. Food Control 2013, 32, 49-54 8. Pleadin, J.; Kovačević, D.; Peršia, N. Ochratoxin A contamination of the autochthonous dry-cured meat product “Slavonski Kulen” during a six-month production process. Food Control 2015, 57, 377-384 9. Cavret, S.; Lecoeura, S. Fusariotoxin transfer in animal. Food and Chemical Toxicology 2006, 44(3), 444-453 10. Pleadin, J.; Staver, M.M.; Vahčić, N.; Kovačević, D.; Milone, S.; LaraSaftićb Scortichini, G. Survey of aflatoxin B1 and ochratoxin A occurrence in traditional meat products coming from Croatian households and markets. Food Control 2015, 52, 71-77 11. Creppy, E.E. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicol Lett. 2002, 28, 127(1-3), 19-28. doi: 10.1016/ s0378-4274(01)00479-9. 12. Speijers, G.J.; Speijers, M.H. Combined toxic effects of mycotoxins. Toxicol Lett. 2004, 153(1), 91-8. doi: 10.1016/j.toxlet.2004.04.046. 13. Fleurat-Lessard, F. Integrated management of risk of stored grain spoilage by seedborne fungi and contamination by storage mould Mycotoxins: An update. Journal of Stored Products Research 2017, 71, 22: 40. 14. Pleadin, J.; Frece, J.; Markov, K. Mycotoxins in food and feed. Adv Food Nutr Res. 2019, 89, 297-345. doi: 10.1016/bs.afnr.2019.02.007. 15. Ringot, D.; Chango, A.; Schneiderb, Y.J.; Larondelle, Y. Toxicokinetics and toxicodynamics of ochratoxin A, an update. Chemico-Biological Interactions 2006, 159, 18-46 16. Sorrenti, V.; Di Giacomo, C.; Acquaviva, R.; Barbagallo, I.; Bognanno, M.; Galvano, F. Toxicity of ochratoxin A and its modulation by antioxidants: a review. Toxins 2013, 5, 1742-1766. 17. Kuroda, K.; Hibi, D.; Ishii, Y.; Yokoo, Y.; Takasu, S.; Kijima, A.; Matsushita K.; Masumura K.I.; Kodama Y.; Yanai T.; Sakai H.; Nohmi T.; Ogawa, K.; Umemura, T. Role of p53 in the progression from ochratoxin A-induced DNA damage to gene mutations in the kidneys of mice. Toxicological Sciences 2015, 144, 65-76 18. Hamid, A.S.; Tesfamariam, I.G.; Zhang, Y.; Zhang, Z.G. Aflatoxin B1-induced hepatocellular carcinoma in developing countries: geographical distribution, mechanism of action and prevention. Oncology letters 2013, 5, 1087-1092 19. McLean, M.; Dutton, M.F. Cellular interactions and metabolism of aflatoxin: an update. Pharmacology and Therapeutics 1995, 65, 163-192. 20. Carter, C.J.; Cannon, M. Structural requirements for the inhibitory action of 12,13-epoxytrichothecenes on protein synthesis in eukaryotes. Biochemical Journal 1977, 166, 399–409. 21. McLaughlin, C.S.; Vaughan, M.H.; Campbell, I.M.; Wei, C.M.; Stafford, M.E.; Hansen, B.S. 1977. Inhibition of protein synthesis by trichothecenes. In: Rodricks, JV.; Hesseltine, CW.; Mehlman, MA. (Eds.), Mycotoxins in Human and Animal Health. Pathotox Publications, Park Forest South, IL, 1997; 263–273. 22. Yang, L.; Tu, D.; Zhao, Z.; Cui, J. Cytotoxicity and apoptosis induced by mixed mycotoxins (T-2 and HT-2 toxin) on primary hepatocytes of broilers in vitro. Toxicon 2017, 129, 1–10. 10.1016/j.toxicon.2017.01.001 23. Bin-Umer, M.A.; McLaughlin, J.E.; Basu, D.; McCormick, S.; Tumer, N.E. Trichothecene mycotoxins inhibit mitochondrial translation--implication for the mechanism of toxicity. Toxins 2011, 3(12), 1484-501. doi: 10.3390/toxins3121484. 24. Pestka, J.J.; Zhou, H.R.; Moon, Y.; Chung, Y.J. Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox. Toxicology Letters 2004,153, 61-73.

25. Weidner, M.; Welsch, T.; Hübner, F.; Schwerdt, G.; Gekle, M.; Humpf, H.U. Identification and apoptotic potential of T-2 toxin metabolites in human cells. Journal of Agricultural and Food Chemistry 2012, 60, 5676-5684. 26. Thiel, P.G. A molecular mechanism for the toxic action of moniliformin, a mycotoxin produced by Fusarium moniliforme. Biochem Pharmacol. 1978, 27, 483-486. 27. Zhang, A.; Cao, J.L.; Yang, B.; Chen, J.H. Zhang, Z.-T.; Li, S.-Y.; Fu, Q.; Hugnes, C.; Caterson, B. Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin-Beck disease. J. Zhejiang Univ. Sci. B. 2010, 11 (3), 200–208. 28. Wang, Y.; Zheng, W.; Bian, X.; Yuan, Y.; Gu, J.; Liu, X.; Liu, Z.; Bian, J. Zearalenone induces apoptosis and cytoprotective autophagy in primary leydig cells. Toxicology Letters 2014, 226 (2), 182–91. 29. Liu, X.; Fan, L.; Yin, S.; Chen, H.; Hu, H. Molecular mechanisms of fumonisin B1-induced toxicities and its applications in the mechanism-based interventions. Toxicon. 2019, 167, 1-5. doi: 10.1016/j.Toxicon.2019.06.009. 30. Brunton, L.L.; Lazo, J.S.; Parker, K.L.; Goodman and Gilman’s. The Pharmacological Basis of Therapeutics. 11th edition. Ed McGraw-Hill. New York, 2006; 1984. 31. EFSA Panel on Contaminants in the Food Chain (CONTAM); Scientific Opinion on Ergot alkaloids in food and feed. EFSA Journal 2012, 10(7), 2798. [158 pp.] doi:10.2903/j.efsa.2012.2798. 32. Forth, W.; Henschler, D.; Rummel, W. Allgemeine und spezielle Pharmakologie und Toxikologie. Urban & Fischer Verlag, München, Germany, 2009; 10. 33. Bbosa, G.S.; Kitya, D.; Odda, J.; Ogwal-Okang, J. Aflatoxin metabolism, effect of epigenetic mechanisms and their role in carcinogenesis. Health 2013, 5, 14-34 34. Food and Agriculture Organisation/World Health Organization, Safety evaluation of certain contaminants in food. Prepared by the Seventy-Second Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). WHO Food Additives Series 2011, 63. 35. Muhammad, I.; Sun, X.; Wang, H.; Li, W.; Wang, X.; Cheng, P.; Li, S., Zhang, X., Hamid, S. Curcumin successfully inhibited the computationally identified CYP2A6 enzyme-mediated bioactivation of aflatoxin B1 in arbor acres broiler. Front. Pharmacol 2017, 8, 143. 10.3389/ fphar.2017.00143 36. Nogaim, Q.A. Aflatoxins M1 and M2 in dairy products. J. Appl. Chem. 2014, 2(5), 14-25. 37. IARC IARC monographs on the evaluation of carcinogenic risks to humans. Iarc Monogr. Eval. Carcinog. Risks Hum. 2010, 93, 9–38. doi: 10.1136/jcp.48.7.691-a. [CrossRef] [Google Scholar] 38. Razzaghi-Abyaneh, M.; Saberi, R.; Sharifan, A.; Rezaee, M.B.; Seifili, R.; Hosseini, S.I.; Shams-Ghahfarokhi, M.; Nikkhah, M.; Saberi, I.; Amani, A. Effects of Heracleum persicum ethyl acetate extract on the growth, hyphal ultrastructure and aflatoxin biosynthesis in Aspergillus parasiticus. Mycotoxin Res. 2013, 29(4), 261-9. doi: 10.1007/s12550-013-0171-1. Epub 2013 Jun 19. PMID: 23780853. 39. Radostits, O.M.; Gay C.C.; Hinchcliff, K.W.; Constable, P.D. A Textbook of the Disease of Cattle, Horses, Sheep, Pigs and Goats. Vet. Med. 2007, 1452–1461. 40. Rustemeyer, S.M.; Lamberson, W.R.; Ledoux, D.R.; Wells, K.; Austin, K.J.; Cammack, K.M. Effects of dietary aflatoxin on the hepatic expression of apoptosis genes in growing barrows. J. Anim. Sci. 2011, 89,916–925. doi: 10.2527/jas.2010-3473. 41. Shi, F.; Seng, X.; Tang, H.; Zhao, S.; Deng, Y.; Jin, R.; Li, Y. Effect of low levels of aflatoxin B1 on performance, serum biochemistry, hepatocyte apoptosis and liver histopathological changes of cherry valley ducks. J. Anim. Vet. Adv. 2013,12,1126–1130. doi: 10.3923/javaa.2013.1126.1130. 42. Monson, M.S.; Settlage, R.E.; McMahon, K.W.; Mendoza, K.M.; Rawal, S.; El-Nezami H.S.; Coulombe, R.A.; Reed, K.M. Response of the hepatic transcriptome to aflatoxin b1in domestic turkey (Meleagris gallopavo) PLoS ONE 2014, 9: e100930. doi: 10.1371/journal.pone.0100930. 43. Hasheminya, S.M.; J, Dehghannya. Strategies for decreasing aflatoxin in livestock feed and milk. Int. Res. J. Appl. Basic Sci. 2013, 4, 1506–1510. 44. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans Chemical agents and related occupations. Iarc Monogr. Eval. Carcinog. Risks Hum. 2012, 100, 9–562. 45. Jafari, T.; Fallah, A.A.; Kheiri, S.; Fadaei, A.; Amini, S.A. Aflatoxin M1 in human breast milk in Shahrekord, Iran and association with dietary factors. Food Addit. Contam. Part B Surveill. 2017, 10, 128–136. doi: 10.1080/19393210.2017.1282545. 46. Marchese, S.; Sorice, A.; Ariano, A.; Florio, S.; Budillon, A.; Costantini, S.; Severino, L. Evaluation of Aflatoxin M1 Effects on the Metabolomic and Cytokinomic Profiling of a Hepatoblastoma Cell Line. Toxins 2018, 10, 436. doi: 10.3390/toxins10110436.

47. Shuib, N.S.; Makahleh, A.; Salhimi, S.M.; Saad, B. Natural occurrence of aflatoxin M1 in fresh cow milk and human milk in Penang, Malaysia. Food Control, 2017, 73, 966–970. doi: 10.1016/j.foodcont.2016.10.013. 48. Tahoun, A.; Ahmed, M.; Abou Elez, R.; AbdEllatif, S. Aflatoxin M1 in Milk and some Dairy Products: Level, Effect of Manufature and Public Health Concerns. Zagazig Vet. J. 2017, 45, 188–196. doi: 10.21608/zvjz.2017.7891. 49. Khan, W.A.; Khan, M.Z.; Khan, A.; Hussan, Z.U.; Saleemi, M.K. Potential of amelioration for aflatoxin B1induced immunotoxic effects in progeny of white leghorn breeder hens co-exposed to E. Journal of Immunotoxicol. 2014, 11, 116-125. 50. Williams, J.H.; Phillips, T.D.; Jolly, P.E.; Stiles, J.K.; Jolly, C.M.; Aggarwal, D. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr. 2004, 80(5),1106-22. doi: 10.1093/ajcn/80.5.1106. PMID: 15531656. 51. Monson, M.; Coulombe, R.; Reed, K. Aflatoxicosis: Lessons from Toxicity and Responses to Aflatoxin B1 in Poultry. Agriculture 2015, 5, 742–777. doi: 10.3390/agriculture5030742. 52. Santacroce, M.P.; Conversano, M.C.; Casalino, E.; Lai, O.; Zizzadoro, C.; Centoducati, G.; Crescenzo, G. Aflatoxins in aquatic species: Metabolism, toxicity and perspectives. Rev. Fish Biol. Fish. 2008, 18, 99–130. doi: 10.1007/s11160-007-9064-8. 53. Dohnal, V.; Wu, Q.; Kuča K. Metabolism of aflatoxins: Key enzymes and interindividual as well as interspecies differences. Arch. Toxicol. 2014, 88, 1635–1644. doi: 10.1007/s00204-014-1312-9. 54. Kuilman, M.E.M.; Maas, R.F.M.; Judah, D.J.; Fink-Gremmels, J. Bovine Hepatic Metabolism of Aflatoxin B1. J. Agric. Food Chem. 1998, 46, 2707–2713. doi: 10.1021/jf980062x. 55. Wogan, G.N.; Kensler, T.W.; Groopman, J.D. Present and future directions of translational research on aflatoxin and hepatocellular carcinoma. A review. Food Addit. Contam. Part A. 2012, 29, 249–257. doi: 10.1080/19440049.2011.563370. 56. Mohd-Redzwan, S.; Jamaluddin, R.; Mutalib, A.; Sokhini, M.; Ahmad, Z. A mini review on aflatoxin exposure in Malaysia: past, present and future. Front. Microbiol. 2013, 4, 334. 10.3389/fmicb.2013.00334. 57. Diaz, D.E.; Hagler, J.W.M.; Blackwelder, J.T.; Eve, J.A.; Hopkins, B.A.; Anderson, K.L.; Jones, F.T.; Whitlow, L.W. Aflatoxin binders II: Reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed. Mycopathologia 2004, 157, 233–241. 58. European Food Safety Authority (EFSA) Opinion of the scientific panel on contaminants in the food chain on a request from the Commission related to aflatoxin B1 as undesirable substance in animal feed. EFSA J. 2004, 39, 1–27. doi: 10.2903/j.efsa.2004. 59. Kaplan, N.M.; Biff, F.; Palmer Sanjay, G.; Revankar. Clinical Implications of Mycotoxins and Stachybotrys. The American Journal of the Medical Sciences 2003, 325 (5), 262-274. 60. Afsah-Hejri, L.; Jinap, S.; Hajeb, P.; Radu, S.; Shakibazadeh, Sh. A Review on Mycotoxins in Food and Feed: Malaysia Case Study. Comprehensive Reviews in. Food Science and Food Safety 2013, 12. doi: 10.1111/1541-4337.12029 61. Elgioushy, M.M.; Elgaml, S.A.; El-Adl, M.M.; Hegazy, A.M.; Hashish, E.A. Aflatoxicosis in cattle: clinical findings and biochemical alterations. Environ Sci Pollut Res Int. 2020 27(28), 35526-35534. doi: 10.1007/s11356-020-09489-3. 62. McKenzie, R.A.; Blaney, B.J.; Connole, M.D.; Fitzpatrick, LA. Acute aflatoxicosis in calves fed peanut hay. Aust Vet J. 1981, 57(6), 284-6. doi: 10.1111/j.1751-0813.1981.tb05816.x. 63. Vaid, J.; Dawra, R.K.; Sharma, O.P.; Negi, S.S. Chronic aflatoxicosis in cattle. Vet Hum Toxicol. 1981, 23(6), 436-8. 64. Jones, F.T.; Genter, M.B.; Hagler, W.M.; Hansen, J.A.; Mowrey, BA.; Poore, M.H.; Whitlow, L.W. Understanding and Coping with Effects of Mycotoxins in Livestock Feed and Forage. North Carolina Cooperative Extension Service, Carolina, 1994; 1-14. 65. Yalagod, S.G.; Mundas, S.; Rao D.G.K.; Tikare, V.; Shridhar, N.B. Histopathological changes in pigs exposed to aflatoxin B1 during pregnancy.. Indian Journal of Animal research 2013, 47(5), 386-391. 66. Ketterer, P.J.; Blaney, B.J.; Moore, C.J.; McInnes, I.S.; Cook, PW. Field cases of aflatoxicosis in pigs. Aust Vet J. 1982, 59(4), 113-7. doi: 10.1111/j.1751-0813.1982.tb02743.x. 67. Dönmez, N.; Dönmez, H.H.; Keskin, E.; Kısadere, İ. Effects of aflatoxin on some haematological parameters and protective effectiveness of esterified glucomannan in Merino rams. Scientific World Journal 2012, 342468. doi: 10.1100/2012/342468. 68. Colakoglu, F.; Donmez, H.H. Effects of aflatoxin on liver and protective effectiveness of esterified glucomannan in Merino rams. Scientific World Journal 2012, 462925. doi: 10.1100/2012/462925. 69. Smith, E.E.; Kubena, L.F.; Braithwaite, CE.; Harvey, RB.; Phillips, TD.; Reine, AH. Toxicological evaluation of aflatoxin and cyclopiazonic acid in broiler chickens. Poult Sci. 1992, 71(7), 1136-44. doi: 10.3382/ps.0711136.

70. Ahmed, M.A.E.; Ravikanth, K.; Rekhe, D.S. Maini,Histopathological alterations in Aflatoxicity and its amelioration with herbomineral toxin binder in broilers. Veterinary World 2009, 2(10). 71. Kumar, R.; Balachandran C. Histopathological changes in broiler chickens fed afl atoxin and cyclopiazonic acid. Veterinarski Arhiv. 2009, 79 (1), 31-40. 72. Duarte, S.C.; Lino, C.M.; Pena, A. Food safety implications of ochratoxin A in animal-derived food products. Vet J. 2012, 192(3), 286-92. doi: 10.1016/j.tvjl.2011.11.002. 73. Liuzzi, V.C.; Fanelli, F.; Tristezza, M.; Haidukowski, M.; Picardi, E.; Manzari, C.; Lionetti, C.; Grieco, F.; Logrieco, A.F.; Thon, M.R.; Pesole, G.; Mulè G. Transcriptional analysis of Acinetobacter sp. neg1 capable of degrading ochratoxin A. Front. Microbiol. 2017, 7, 2162. 10.3389/ fmicb.2016.02162 74. Ladeira, C.; Frazzoli, C.; Orisakwe, OE. Engaging one health for non-communicable diseases in Africa: perspective for mycotoxins. Front. Public Health 2017, 5, 266. 10.3389/fpubh.2017.00266 75. Russo, P.; Capozzi, V.; Spano, G.; Corbo, M.R.; Sinigaglia, M.; Bevilacqua, A. Metabolites of microbial origin with an impact on health: ochratoxin A and biogenic amines. Front. Microbiol. 2016, 7,482. 10.3389/fmicb.2016.00482 76. EFSA European Food Safety Authority. Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to OTA in food. Question n. efsa-q 2005-154. EFSA J. 2006, 365,1–56. 77. Felizardo, R.J.; Câmara, N.O. Hepatocellular carcinoma and food contamination: aflatoxins and ochratoxin A as a great prompter. World J Gastroenterol. 2013, 19(24), 3723-5. doi: 10.3748/wjg.v19.i24.3723. 78. Fink-Gremmels, J.; Malekinejad, H. Clinical effects and biochemical mechanisms associated with exposure to the mycoestrogen zearalenone. Animal Feed Science and Technology 2007, 137(3-4), 326–41. 79. Biasucci, G.; Calabrese, G.; Di Giuseppe, R.; Carrara, G.; Colombo, F.; Mandelli, B.; Maj, M.; Bertuzzi, T.; Pietri, A.; Rossi, F. The presence of ochratoxin A in cord serum and in human milk and its correspondence with maternal dietary habits. Eur J Nutr. 2010, 50, 211–218 80. Anzai, N.; Jutabha, P.; Endou, H. Molecular mechanism of ochratoxin a transport in the kidney. Toxins (Basel) 2010, 2(6), 1381-98. doi: 10.3390/toxins2061381. Epub 2010 Jun 9. PMID: 22069643; PMCID: PMC3153260. 81. Zlender, V.; Breljak, D.; Ljubojević, M.; Flajs, D.; Balen, D.; Brzica, H.; Domijan, A.M.; Peraica, M.; Fuchs, R.; Anzai, N.; Sabolić, I. Low doses of ochratoxin A upregulate the protein expression of organic anion transporters Oat1, Oat2, Oat3 and Oat5 in rat kidney cortex. Toxicol Appl Pharmacol. 2009, 239(3), 284-96. doi: 10.1016/j.taap.2009.06.008. Epub 2009 Jun 16. PMID: 19538982. 82. Arsani, R.K.; Patial, V.; Thakur, M. Ochratoxin A: Possible Mechanisms of Toxicity. In book: Ochratoxins: Biosynthesis, Detection and Toxicity Publisher: Nova Publishers, New York, pp.57-89. 83. Capei, R.; Pettini, L.; Mandò Tacconi, F. Occurrence of Ochratoxin A in breakfast cereals and sweet snacks in Italy: dietary exposure assessment. Ann Ig. 2019, 31(2), 130-139. doi: 10.7416/ai.2019.2265. PMID: 30714610. 84. el Khoury, A.; Atoui, A. Ochratoxin a: general overview and actual molecular status. Toxins (Basel) 2010, 2(4), 461-93. doi: 10.3390/ toxins2040461. Epub 2010 Mar 29. PMID: 22069596; PMCID: PMC3153212. 85. European Commission. Commission regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union L. 2006, 364, 5-24. 86. Pavlović, N.M. Balkan endemic nephropathy-current status and future perspectives. Clin Kidney J. 2013, 6(3), 257-65. doi: 10.1093/ckj/ sft049. PMID: 26064484; PMCID: PMC4400492. 87. Reddy, K.R.N.; Salleh, B.; Saad, B.; Abbas, H.K. An overview of mycotoxin contamination in foods and its implications for human health. Toxin Reviews. 2010, 29(1), 3-26 88. Hassen, W.; Abid-Essafi, S.; Achour, A.; Guezzah, N.; Zakhama, A.; Ellouz, F.; Creppy, E.E.; Bacha, H. Karyomegaly of tubular kidney cells in human chronic interstitial nephropathy in Tunisia: respective role of Ochratoxin A and possible genetic predisposition. Hum Exp Toxicol. 2004, 23(7), 339-46. doi: 10.1191/0960327104ht458oa. PMID: 15311851. 89. O’Brien, E.; Dietrich, D.R. Ochratoxin A: the continuing enigma. Crit Rev Toxicol. 2005, 35(1), 33-60. doi: 10.1080/10408440590905948. PMID: 15742902. 90. Yordanova, P.; Wilfried, K.; Tsolova, S.; Dimitrov, P. Ochratoxin A and β2-microglobulin in BEN patients and controls. Toxins (Basel) 2010, 2(4), 780-92. doi: 10.3390/toxins2040780. Epub 2010 Apr 20. PMID: 22069610; PMCID: PMC3153209.

91. Sauvant, C.; Holzinger, H.; Gekle, M. The nephrotoxin ochratoxin A induces key parameters of chronic interstitial nephropathy in renal proximal tubular cells. Cell Physiol Biochem. 2005, 15(1-4), 125-34. doi: 10.1159/000083660. PMID: 15665523. 92. Stoev, S.D.; Hald, B.; Mantle, P. Porcine nephropathy in Bulgaria: a progressive syndrome of complex of uncertain (mycotoxin) etiology. Vet Rec. 1998, 142, 190–194. 93. Perši, N.; Pleadin, J.; Kovačević, D.; Scortichini, G.; Milone, S. Ochratoxin A in raw materials and cooked meat products made from OTA-treated pigs. Meat Sci. 2014, 96(1), 203-10. doi: 10.1016/j.meatsci.2013.07.005. Epub 2013 Jul 12. PMID: 23906754. 94. Gresham, A.; Done, S.; Livesey, C.; MacDonald, S.; Chan, D.; Sayers, R.; Clark, C.; Kemp, P. Survey of pigs’ kidneys with lesions consistent with PMWS and PDNS and ochratoxicosis. Part 2: pathological and histological findings. Vet Rec. 2006, 159(23), 761-8. PMID: 17142623. 95. Cook, W.O.; Osweiler, G.D.; Anderson, T.D.; Richard, JL. Ochratoxicosis in Iowa swine. J Am Vet Med Assoc. 1986, 188(12), 1399-402. PMID: 3744966. 96. Patial, V.; Asrani, R.K.; Patil, R.D.; Ledoux, D.R.; Rottinghaus, G.E. Pathology of ochratoxin A-induced nephrotoxicity in Japanese quail and its protection by sea buckthorn (Hippophae rhamnoides L.). Avian Dis. 2013, 57(4), 767-79. doi: 10.1637/10549-040913-Reg.1. PMID: 24597120. 97. Patial, V.; Asrani, R.K.; Patil, R.D.; Kumar, N. Protective Effect of Sea buckthorn (Hippophae rhamnoides L.) Leaves on Ochratoxin-A Induced Hepatic Injury in Japanese quail. Veterinary Research International 2015, 3(4), 98-108. 98. Patil, R.D.; Dwivedi, P.; Sharma, A.K. Critical period and minimum single oral dose of ochratoxin A for inducing developmental toxicity in pregnant Wistar rats. Reprod Toxicol. 2006, 22(4), 679-87. doi: 10.1016/j.reprotox.2006.04.022. Epub 2006 Jun 14. PMID: 16781114. 99. Mazzoni, E.; Scandolara, A.; Giorni, P.; Pietri, A.; Battilani, P. Field control of Fusarium ear rot, Ostrinia nubilalis (Hübner), and fumonisins in maize kernels. Pest Manag Sci. 2011, 67(4):458-65. doi: 10.1002/ps.2084. Epub 2011 Jan 6. PMID: 21394878. 100. Lerda, D. Fumonisins in foods from Cordoba (Argentina), presence: mini review. Toxicol. 2017, 3, 125 10.4172/2476-2067.1000125 101. Lumsangkul, C.; Chiang, HI.; Lo, NW.; Fan, YK.; Ju, JC. Developmental Toxicity of Mycotoxin Fumonisin B₁ in Animal Embryogenesis: An Overview. Toxins (Basel) 2019, 11(2), 114. doi: 10.3390/toxins11020114. PMID: 30781891; PMCID: PMC6410136. 102. Liu, X.; Fan, L.; Yin, S.; Chen, H.; Hu, H. Molecular mechanisms of fumonisin B1-induced toxicities and its applications in the mechanism-based interventions. Toxicon. 2019, 167, 1-5. doi: 10.1016/j.toxicon.2019.06.009. Epub 2019 Jun 4. PMID: 31173793. 103. da Rocha, M.E.B.; Freire, F.D.C.O.; Maia, F.E.F.; Guedes, M.I.F.; Rondina, D. Mycotoxins and their effects on human and animal health. Food Control 2014, 36, 159–165. 10.1016/j.foodcont.2013.08.021 104. IARC (International Agency for Research on Cancer). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 82. Lyon, International Agency for Research on Cancer, 2002; 82. 105. WHO (World Health Organization). Safety Evaluation of Certain Mycotoxins in Food. WHO Food Additive Series 47, Geneva, 2001. 106. Abnet, C.C.; Borkowf, C.B.; Qiao, Y.L.; Albert, P.S.; Wang, E.; Merrill AH, Jr.; Mark, S.D.; Dong, Z.W.; Taylor, P.R.; Dawsey, S.M. Sphingolipids as biomarkers of fumonisin exposure and risk of esophageal squamous cell carcinoma in china. Cancer Causes Control 2001, 12(9), 821-8. doi: 10.1023/a:1012228000014. PMID: 11714110. 107. Voss, K.A.; Riley, T.R.; Waes Gelineau-van, J. Fumonisin B1 induced neural tube defects were not increased in LM/Bc mice fed folate-deficient diet. Molecular Nutrition & Food Research, 2014, 58(6). 108. Sandmeyer, L.S.; Vujanovic, V.; Petrie, L.; Campbell, J.R.; Bauer, B.S.; Allen, AL.; Grahn, B.H. Optic neuropathy in a herd of beef cattle in Alberta associated with consumption of moldy corn. Can Vet J. 2015, 56(3), 249-56. PMID: 25750444; PMCID: PMC4327135. 109. Mathur, S.; Constable, P.D.; Eppley, R.M.; Waggoner, A.L.; Tumbleson, M.E.; Haschek, W.M. Fumonisin B(1) is hepatotoxic and nephrotoxic in milk-fed calves. Toxicol Sci. 2001, 60(2), 385-96. doi: 10.1093/toxsci/60.2.385. PMID: 11248152. 110. Wilson, T.M.; Ross, P.F.; Rice, L.G.; Osweiler, G.D.; Nelson, H.A.; Owens, D.L.; Plattner, R.D.; Reggiardo, C.; Noon, TH.; Pickrell, JW. Fumonisin B, levels associated with an epizootic of equine leukoencephalomalacia. J Vet Diagn Invest. 1990, 2, 213-6. 111. Haschek, W.M.; Gumprecht, L.A.; Smith, G.; Tumbleson, M.E.; Constable, P.D. Fumonisin toxicosis in swine: an overview of porcine pulmonary edema and current perspectives. Environ Health Perspect. 2001,109 (Suppl 2), 251-7. doi: 10.1289/ehp.01109s2251. PMID: 11359693; PMCID: PMC1240673. 112. Osweiler, G.D.; Ross, P.F.; Wilson, T.M.; Nelson, P.E.; Witte, S.T.; Carson, T.L.; Rice, L.G.; Nelson, H.A. Characterization of an epizootic of pulmonary edema in swine associated with fumonisin in corn screenings. J. Vet. Diagn. Invest. 1992, 4, 53–59. 113. Colvin, B.M.; Cooley, A.J.; Beaver, RW. Fumonisin toxicosis in swine: clinical and pathological findings. J. Vet. Diagn. Invest. 1993, 5, 232–241.

114. Giannitti, F.; Diab, S.; Pacin, A.; Barrandeguy, M.; Larrere, C et al. Equine leukoencephalomalacia due to fumonisins B1 and B2 in Argentina. Pesq Vet Bras. 2011, 31, 407-412. 115. Kovacić, S.; Pepeljnjak, S.; Petrinec, Z.; Klarić, M.S. Fumonisin B1 neurotoxicity in young carp (Cyprinus carpio L.). Arh Hig Rada Toksikol. 2009, 60(4), 419-26. doi: 10.2478/10004-1254-60-2009-1974. PMID: 20061242. 116. Benlasher, E.; Geng, X.; Nguyen, N.T.; Tardieu, D.; Bailly, J.D.; Auvergne, A.; Guerre, P. Comparative effects of fumonisins on sphingolipid metabolism and toxicity in ducks and turkeys. Avian Dis. 2012, 56(1), 120-7. doi: 10.1637/9853-071911-Reg.1. PMID: 22545537. 117. Ledoux, D.R.; Brown, T.P.; Weibking, TS.; Rottinghaus, GE. Fumonisin toxicity in broiler chicks. J Vet Diagn Invest. 1992, 4(3), 330-3. doi: 10.1177/104063879200400317. PMID: 1515495. 118. Deshmukh, S.; Asrani, R.K.; Jindal, N.; Ledoux, D.R.; Rottinghaus, G.E.; Sharma, M.; Singh, S.P. Effects of Fusarium moniliforme culture material containing known levels of fumonisin B1 on progress of Salmonella Gallinarum infection in Japanese quail: clinical signs and hematologic studies. Avian Dis. 2005, 49(2), 274-80. doi: 10.1637/7296-102804R. PMID: 16094834. 119. Deshmukh, S.; Asrani, R.K.; Ledoux, D.R.; Rottinghaus, G.E.; Bermudez, A.J.; Gupta, V.K. Pathologic changes in extrahepatic organs and agglutinin response to Salmonella Gallinarum infection in Japanese quail fed Fusarium verticillioides culture material containing known levels of fumonisin B1. Avian Dis. 2007, 51(3):705-12. doi: 10.1637/0005-2086(2007)51 120. Jaradat, Z.W, T-2 mycotoxin in the diet and its effects on tissues. In: Watson RR and Preedy VR. Reviews in Food and Nutrition Toxicity 2005, 4, 173-212. 121. SCF, Scientific Committee on Food. Opinion of the Scientific Committee on Food on Fusarium Toxins. Part 5: T-2 Toxin and HT-2 Toxin. 2001, SCF/CS/CNTM/MYC/25 Rev 6 Final. http://ec.europa.eu/food/fs/sc/scf/out88_en.pdf. 122. Nathanail, A.V.; Syvähuoko, J.; Malachová, A.; Jestoi, M.; Varga, E.; Michlmayr, H.; Adam, G.; Sieviläinen, E.; Berthiller, F.; Peltonen, K. Simultaneous determination of major type A and B trichothecenes, zearalenone and certain modified metabolites in Finnish cereal grains with a novel liquid chromatography-tandem mass spectrometric method. Anal Bioanal Chem. 2015, 407(16), 4745-55. doi: 10.1007/s00216-015-86764. Epub 2015 May 3. PMID: 25935671; PMCID: PMC4446524. 123. Adhikari, M.; Negi, B.; Kaushik, N.; Adhikari, A.; Al-Khedhairy, A.A.; Kaushik, N.K.; Choi, E.H. T-2 mycotoxin: toxicological effects and decontamination strategies. Oncotarget 2017, 8(20), 33933-33952. doi: 10.18632/oncotarget.15422. PMID: 28430618; PMCID: PMC5464924. 124. Borutova, R.; Faix, S.; Placha, I.; Gresakova, L.; Cobanova, K.; Leng, L. Effects of deoxynivalenol and zearalenone on oxidative stress and blood phagocytic activity in broilers. Arch Anim Nutr. 2008 62(4), 303-12. doi: 10.1080/17450390802190292. PMID: 18763624 125. Eriksen, G.S.; Petterson, H. Toxicological evaluation of trichothecenes in animal feed. Animal Feed Science and Technology 2004, 114, 205–239DOI:10.1016/J.ANIFEEDSCI.2003.08.008 Corpus ID: 85123463 126. Kolf-Clauw, M.; Sassahara, M.; Lucioli, J.; Rubira-Gerez, J.; Alassane-Kpembi, I.; Lyazhri, F.; Borin, C.; Oswald, I.P. The emerging mycotoxin, enniatin B1, down-modulates the gastrointestinal toxicity of T-2 toxin in vitro on intestinal epithelial cells and ex vivo on intestinal explants. Arch Toxicol. 2013, 87(12), 2233-41. doi: 10.1007/s00204-013-1067-8. Epub 2013 May 7. PMID: 23649843. 127. Alizadeh, A.; Braber, S.; Akbari, P.; Garssen, J.; Fink-Gremmels, J. Deoxynivalenol Impairs Weight Gain and Affects Markers of Gut Health after Low-Dose, Short-Term Exposure of Growing Pigs. Toxins (Basel) 2015, 7(6), 2071-95. doi: 10.3390/toxins7062071. PMID: 26067367; PMCID: PMC4488690. 128. Zhu, Y.; Hassan, Y.I.; Shao, S.; Zhou, T. Employing immuno-affinity for the analysis of various microbial metabolites of the mycotoxin deoxynivalenol. J Chromatogr A. 2018, 1556, 81-87. doi: 10.1016/j.chroma.2018.04.067. Epub 2018 May 1. PMID: 29731291. 129. Ingalls, J.R. Influence of deoxynivalenol on feed consumption by dairy cows. Anim. Feed Sci. Technol. 1996, 60, 297-300. ISSN 0377-8401 130. Cote, L. M.; Dahlem, A. M.; Yoshizawa, T.; Swanson, S. P.; Buck, W. B. Excretion of deoxynivalenol and its metabolites in milk, urine, and feces of lactating dairy cows. Journal of Dairy Science 1986, 69, 2416–2423. 131. Hendry, K.M.; Cole, EC. A review of mycotoxins in indoor air. J. Toxicol. Environ. Health Sci. 1993, 38, 183–198. doi: 10.1080/15287399309531711. 132. Weindenborner, M. Natural Mycotoxin Contamination in Humans and Animals. Springer, Switzerland, 2015. 133. Zouagui, Z.; Asrar, M.; Lakhdissi, H.; Abdennebi, E. Prevention of mycotoxin effects in dairy cows by adding an anti-mycotoxin product in feed. J. Mater. Environ. Sci. 2017, 8, 3766–3770. [Google Scholar]

134. Valgaeren, B.; Théron, L.; Croubels, S.; Devreese, M.; De Baere, S.; Van Pamel, E.; Daeseleire, E.; De Boevre, M.; De Saeger, S.; Vidal, A.; Di Mavungu, J.D.; Fruhmann, P.; Adam, G.; Callebaut, A.; Bayrou, C.; Frisée, V.; Rao, A.S.; Knapp, E.; Sartelet, A.; Pardon, B.; Deprez, P.; Antonissen, G. The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics. Arch Toxicol. 2019, 93(2), 293-310. doi: 10.1007/s00204-018-2368-8. Epub 2018 Dec 10. PMID: 30535711. 135. Helferich, W.G.; Garrett, WN.; Hsieh, DPH.; Baldwin, RL. Feedlot performance and tissue residues of cattle consuming diets containing aflatoxins. J Anim Sci. 1986, 62, 691–696. pmid:3700268 136. Petrie, L.; Robb, J.; Stewart, A.F. The identification of T-2 toxin and its association with a haemorrhagic syndrome in cattle. Vet Rec. 1977, 101, 326–326. pmid:929903 137. Wannemacher, R.W.; Brunner, D.L.; Neufeld, H.A. Toxicity of trichothecenes and other related mycotoxins in laboratory animals. In: Smith J.E. and Henderson R.S. (Eds.), “Mycotoxins and Animal Foods.” CRC Press, Inc., Boca Raton FL, 1991. pp. 499–552. 138. Serviento, A.M.; Brossard, L.; Renaudeau, D. An acute challenge with a deoxynivalenol-contaminated diet has short- and long-term effects on performance and feeding behavior in finishing pigs. J Anim Sci. 2018, 96(12), 5209-5221. doi: 10.1093/jas/sky378. PMID: 30423126; PMCID: PMC6276570. 139. Bracarense, A.P.F.L.; Lucioli, J.; Grenier, B.; Pacheco, G.D.; Moll, W-D.; Schatzmayr, G.; Oswald, I.P. Chronic ingestion of deoxynivalenol and fumonisin, alone or in interaction, induces morphological and immunological changes in the intestine of piglets. Brit J Nutr. 2012, 107, 1776–1786. 140. Gerez,J.R.; Pinton, P.; Callu, P.; Grosjean, F.; Oswald, IP.; Bracarense, APFL. Deoxynivalenol alone or in combination with nivalenol and zearalenone induce systemic histological changes in pigs. Exp Toxicol Pathol. 2015, 67, 89–98. 141. Gerez, J.R.; Desto, S.S.; Bracarense, A.P.F.R.L. Deoxynivalenol induces toxic effects in the ovaries of pigs: An ex vivo approach. Theriogenology 2017, 90, 94-100. doi: 10.1016/j.theriogenology.2016.10.023. Epub 2016 Nov 9. PMID: 28166994. 142. Ferreras, M.C.; Benavides, J.; García-Pariente, C.; Delgado, L.; Fuertes, M.; Muñoz, M.; García-Marín, J.F.; Pérez, V. Acute and chronic disease associated with naturally occurring T-2 mycotoxicosis in sheep. J Comp Pathol. 2013, 148(2-3):236-42. doi: 10.1016/j.jcpa.2012.05.016. Epub 2012 Jul 20. PMID: 22819015. 143. Nayakwadi, S.; Ramu, R.; Kumar Sharma, A.; Kumar Gupta, V.; Rajukumar, K.; Kumar, V.; Shirahatti, P.S.; Rashmi, L.; Basalingappa, K.M. Toxicopathological studies on the effects of T-2 mycotoxin and their interaction in juvenile goats. PLoS One, 2020, 26, 15(3), e0229463. doi: 10.1371/journal.pone.0229463. PMID: 32214355; PMCID: PMC7098593. 144. Richard, J.L.; Payne, G.A.; Desjardins, AE.; Maragos, C.; Norred, W.; Pestka, J. Mycotoxins: Risks in plant, animal and human systems. CAST Task Force Report 2003,139, 101–3. 145. Bennet, JW.; Klich, M. Mycotoxins. Clinical microbiology review, 2003, 16(3), 497–516. DOI: 10.1128/CMR.16.3.497-516.2003 146. Yumbe-Guevara, B.; Imoto, T.; Yoshizawa, T. Effects of heating procedures on deoxynivalenol, nivalenol and zearalenone levels in naturally contaminated barley and wheat. Food Additives and Contaminants 2003, 20 (12), 1132–40. doi: Crossref. 147. Polak, M.; Paluszewski A.; Rybarczyk, L.; Gajęcki, M. Influence of zearalenone micotoxicosis on selected immunological, haematological and biochemical indexes of blood plasma in bitches. Polish Journal of Veterinary Sciences 2004, 7 (3), 175–80. doi: Crossref 148. Zwierzchowski, W.; Przybyłowicz, M.; Obremski, K.; Zielonka, L.; Skorska-Wyszyńska, E.; Gajecka, M., Polak, M.; Jakimiuk, E.; Jana, B.; Rybarczyk, L et al. Level of zearalenone in blood serum and lesions in ovarian follicles of sexually immature gilts in the course of zearalenone micotoxicosis. Polish Journal of Veterinary Sciences 2005, 8 (3), 209–18. 149. Rai, A.; Das, M.; Tripathi, A. Occurrence and toxicity of a fusarium mycotoxin, zearalenone. Crit Rev Food Sci Nutr. 2020, 60(16), 2710-2729. doi: 10.1080/10408398.2019.1655388. Epub 2019 Aug 26. PMID: 31446772. 150. Wang, Y.; Wong, T.Y.; Chan, F.L.; Chen, S.; Leung, L.K. Assessing the effect of food mycotoxins on aromatase by using a cell-based system. Toxicology in Vitro: An International Journal Published in Association with Bibra 2014, 28 (4), 640–6. doi: Crossref. 151. Kuiper-Goodman, T.; Scott, P.M.; Watanabe, H. Risk assessment of the mycotoxin zearalenone. Regul. Toxicol. Pharmacol. 1987, 7, 253–306. 10.1016/0273-2300(87)90037-7 [PubMed] [CrossRef] [Google Scholar] 152. Abbès, S.; Salah-Abbès, J.B.; Ouanes, Z.; Houas, Z.; Othman, O.; Bacha H.; Abdel-Wahhab, M.A.; Oueslati, R. Preventive role of phyllosilicate clay on the immunological and biochemical toxicity of zearalenone in balb/c mice. International Immunopharmacology 2000, 6 (8), 1251–8. doi: Crossref.

153. Abbès, S.; Ouanes, Z.; Salah-Abbès J.B.; Abdel, W.A.; Oueslati, M.R.; Bacha, H. Preventive role of aluminosilicate clay against induction of micronuclei and chromosome aberrations in bone-marrow cells of balb/c mice treated with zearalenone. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2007, 631 (2), 85–92. doi: Crossref. 154. Wang, Y.C.; Deng, J.L.; Xu S.W.; Peng, X.; Zuo, Z.C.; Cui, H.M.; Wang, Y.; Ren, Z.H. Effects of zearalenone on IL-2, IL-6, and IFN-γ mRNA levels in the splenic lymphocytes of chickens. Scientific World Journal 2012, 2012, 567327. doi: 10.1100/2012/567327. Epub 2012 May 2. PMID: 22645433; PMCID: PMC3354442. 155. Murata, H.; Sultana, P.; Shimada, N.; Yoshioka, M. Structure-activity relationships among zearalenone and its derivatives based on bovine neutrophil chemiluminescence. Veterinary and Human Toxicology 2003, 45 (1), 18–20. 156. Poor, M.; Kunsagi-Mate, S.; Sali, N.; Koszegi, T.; Szente ,L.; Peles-Lemli, B. Interactions of zearalenone with native and chemically modified cyclodextrins and their potential utilization. J. Photochem. Photobiol. B. 2015, 151, 63–68. 10.1016/j.jphotobiol.2015.07.009 [PubMed] [CrossRef] [Google Scholar] 157. Fink-Gremmels, J.; Malekinejad, H. Clinical effects and biochemical mechanisms associated with exposure to the mycoestrogen zearalenone. Animal Feed Science and Technology 2007, 137 (3–4, 1), 326-341. 158. Biehl, M.; Prelusky, D.; Koritz, G.; Hartin, K.; Buck, W.; Trenholm, H. Biliary excretion and enterohepatic cycling of zearalenone in immature pigs. Toxicology and Applied Pharmacology 1993, 121 (1), 152–9. doi: Crossref. 159. Sambuu, R.; Takagi, M.; Shiga, S.; Uno, S.; Kokushi, E.; Namula, Z et al. Detection of zearalenone and its metabolites in naturally contaminated porcine follicular fluid by using liquid chromatography-tandem mass spectrometry. J. Reprod. Dev. 2011, 57, 303–306. 10.1262/ jrd.10-106M [PubMed] [CrossRef] [Google Scholar] 160. Cortinovis, C.; Pizzo, F.; Spicer, L.J.; Caloni, F. Fusarium mycotoxins: effects on reproductive function in domestic animals–a review. Theriogenology 2013, 80, 557–564. doi: 10.1016/j.theriogenology.2013.06.018 161. Wang, H.W.; Wang, J.Q.; Zheng, B.Q.; Li ,SL.; Zhang, YD.; Li, FD.; Zheng, N. Cytotoxicity induced by ochratoxin A, zearalenone, and α-zearalenol: effects of individual and combined treatment. Food Chem Toxicol. 2014, 71, 217-24. doi: 10.1016/j.fct.2014.05.032. Epub 2014 Jun 18. PMID: 24952310. 162. EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food, 2011. https://doi.org/10.2903/j.efsa.2011.2407 163. GAIN. China releases standard for maximum levels of mycotoxins in foods (global agriculture information network). (China Food and Drug Administration) CFDA, GAIN report no. CH18026, 2018, 1–10. 164. Massart, F.; Meucci, V.; Saggese, G.; Soldani, G. High growth rate of girls with precocious puberty exposed to estrogenic mycotoxins. J Pediatr. 2008, 152(5), 690-695.e1. doi: 10.1016/j.jpeds.2007.10.020. Epub 2008 Feb 20. PMID: 18410776. 165. Kuciel-Lisieska, G.; Obremski, K.; Stelmachów, J.; Gajecka, M.; Zielonka, Ł.; Jakimiuk, E.; Gajecki, M. Presence of zearalenone in blood plasma in women with neoplastic lesions in the mammary gland. Bulletin of the Veterinary Institute in Pulawy 2008, 52, 671–674. 166. Tomaszewski, J.; Miturski, R.; Semczuk, A.; Kotarski, J.; Jakowicki, J. Tissue zearalenone concentration in normal, hyperplastic and neoplastic human endometrium. Ginekologia Polska. 1998, 69 (5), 363–6. 167. Minervini, F., and Dell0Aquila, M. E. Zearalenone and reproductive function in farm animals. Int. J. Mol. Sci. 2008, 9, 2570–2584. doi: 10.3390/ ijms9122570 168. Zinedine, A.; Soriano, J. M.; Molto, J. C.; and Manes, J. Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: An oestrogenic mycotoxin. Food and Chemical Toxicology 2007, 45 (1), 1–18 169. Weaver, G.A.; Kurtz, H.J.; Behrens, J.C.; Robison, T.S.; Seguin, B.E.; Bates, F.Y et al. Effect of zearalenone on the fertility of virgin dairy heifers. Am. J. Vet. Res. 1986, 47, 1395–1397 170. Obremski, K.; Gajecki, M.; Zwierzchowski, W.; Zielonka, L.; Otrocka-Domagala, I.; Rotkiewicz, T. et al. Influence of zearalenone on reproductive system cell proliferation in gilts. Pol. J. Vet. Sci. 2003, 6, 239–245. 171. Etienne, M.; Dourmad, J.Y. Effects of zearalenone or glucosinolates in the diet on reproduction in sows: A review. Livest. Prod. Sci. 1994, 40, 99–113. doi: 10.1016/0301-6226(94)90040-X 172. Malekinejad, H.; Maas-Bakker, R.; Fink-Gremmels, J. Species differences in the hepatic biotransformation of zearalenone. Vet J. 2006, 172(1), 96-102. doi: 10.1016/j.tvjl.2005.03.004. PMID: 15907386. 173. Zhang, G.L.; Feng, Y.L.; Song, J.L.; Zhou, X.S. Zearalenone: A Mycotoxin with Different Toxic Effect in Domestic and Laboratory Animals’ Granulosa Cells. Front Genet. 2018, 18, 9:667. doi: 10.3389/fgene.2018.00667. PMID: 30619484; PMCID: PMC6305301.

174. Yang, J.Y.; Wang, G.X.; Liu, JL.; Fan, JJ.; Cui, S. Toxic effects of zearalenone and its derivatives α-zearalenol on male reproductive system in mice. Reproductive Toxicology 2007, 24 (3-4), 381–7. doi: Crossref. 175. Ito, Y.; Ohtsubo, K. Effects of neonatal administration of zearalenone on the reproductive physiology of female mice. J. Vet. Med. Sci. 1994, 56, 1155–1159. doi: 10.1292/jvms.56.1155 176. Teixeira, L.C.; Montiani-Ferreira, F.; Dittrich, R.; Santin, E. Effects of zearalenone in prepubertal gilts. Pesquisa Veterinária Brasileira. 2011, 31(8), 656-662 177. Logrieco, A.; Mule, G.; Moretti, A.; Bottalico, A. Toxigenic Fusarium Species and Mycotoxins Associated with Maize Ear Rot in Europe. Eur J Plant Pathol 2002, 108, 597- 609. 178. Desjardins, A. E.; Maragos, C. M.; Proctor, R. H. Maize Ear Rot and Moniliformin Contamination by Cryptic Species of Fusarium subglutinans. J Agric Food Chem 2006, 54, 7383-7390. 179. Kokkonen, M.; Ojala, L.; Parikka, P.; Jestoi, M. Mycotoxin production of selected Fusarium species at different culture conditions. Int J Food Microbiol 2010, 143, 17-25. 180. Hallas-Moeller, M.; Nielsen, K. F.; Frisvad, J. C. Production of the Fusarium Mycotoxin Moniliformin by Penicillium melanoconidium. J Agric Food Chem 2016, 64, 4505-4510. 181. Jonsson, M.; Jestoi, M.; Nathanail, A.V.; Kokkonen, U.M.; Anttila, M.; Koivisto, P.; Karhunen, P.; Peltonen, K. Application of OECD Guideline 423 in assessing the acute oral toxicity of moniliformin. Food Chem Toxicol. 2013, 53, 27-32. doi: 10.1016/j.fct.2012.11.023. Epub 2012 Nov 28. PMID: 23201451. 182. Burmeister, H.; Ciegler, A.; Vesonder, R.F. Moniliformin, a metabolite of Fusarium moniliforme NRRL 6322: purification and toxicity. Appl. Environ. Microbiol. 37, 11–13. 183. Ueno, Y. Developments in food science. Gen. Toxicol. 1983, 4, 135–146. 184. Nagaraj, R.Y.; Wu, W.; Will, J.A.; Vesonder, R.F. Acute cardiotoxicity of moniliformin in broiler chickens as measured by electrocardiography. Avian Dis. 1996, 40, 223–227 185. Kriek, N.P.J.; Marasas, W.F.O.; Steyn, P.S.; Van Rensburg, S.J.; Steyn M.; Toxicity of a moniliformin-producing strain of Fusarium moniliforme var. subglutinans isolated from maize. Food Cosmet. Toxicol. 1977, 15, 579–587 186. Nesic, K.; Ivanovic, S.; Nesic, V. Fusarial toxins: secondary metabolites of Fusarium fungi. Rev Environ Contam Toxicol. 2014, 228, 101-20. doi: 10.1007/978-3-319-01619-1_5. PMID: 24162094. 187. Burka, L.T.; Doran, J.; Wilson, B. J. Enzyme inhibition and the toxic action of moniliformin and other vinylogous α-ketoacids. Biochem Pharmacol. 1982, 31, 79-84. 188. Gathercole, P. S.; Thiel, P. G.; Hofmeyr, J. H. S. Inhibition of pyruvate dehydrogenase complex by moniliformin. Biochem J. 1986, 233, 719-723 189. Cao, J.; Zhang, A.; Yang, B.; Zhang, Z.T.; Fu, Q.; Hughes, C.E.; Caterson, B. The effect of fungal moniliformin toxin and selenium supplementation on cartilage metabolism in vitro. Osteoarthr. Cartil. 2007, 15(Suppl. 3), C108. 190. Jonsson, M.; Atosuo, J.; Jestoi, M.; Nathanail, A.V.; Kokkonen, U.M.; Anttila, M.; Koivisto, P.; Lilius, E.M.; Peltonen, K. Repeated dose 28-day oral toxicity study of moniliformin in rats. Toxicol. Lett. 2015, 233, 38-44. 191. Sharma, D.; Asrani, R.K.; Ledoux, D.R.; Rottinghaus, G.E.; Gupta, V.K. Toxic interaction between fumonisin B1 and moniliformin for cardiac lesions in Japanese quail. Avian Dis. 2012, 56, 545-554. 192. Stoev, S.; Denev, S.; Dutton, M.; Nkosi, B. Cytotoxic Effect of Some Mycotoxins and their Combinations on Human Peripheral Blood Mononuclear Cells as Measured by the MTT Assay. The Open Toxinology Journal, 2009, 2, 1-8 193. Domijan, A.M.; Gajski, G.; Novak Jovanović, I.; Gerić, M.; Garaj-Vrhovac, V. In vitro genotoxicity of mycotoxins ochratoxin A and fumonisin B(1) could be prevented by sodium copper chlorophyllin--implication to their genotoxic mechanism. Food Chem. 2015, 170:455-62. doi: 10.1016/j.foodchem.2014.08.036. Epub 2014 Aug 19. PMID: 25306371. 194. Heussner, A.H.; Bingle, L.E. Comparative Ochratoxin Toxicity: A Review of the Available Data. Toxins (Basel). 2015, 7(10), 4253-82. doi: 10.3390/toxins7104253. PMID: 26506387; PMCID: PMC4626733. 195. Khan, M.A.; Asrani, R.K.; Iqbal, A.; Patil, R.D. Fumonisin B1 and ochratoxin A nephrotoxicity in Japanese quail: An ultrastructural assessment. Comparative Clinical Pathology. 2013, 22(5), 835–843.

196. Klarić, M.S.; Rašić, D.; Peraica, M. Deleterious effects of mycotoxin combinations involving ochratoxin A. Toxins (Basel) 2013, 5(11), 1965-87. doi: 10.3390/toxins5111965. PMID: 24189375; PMCID: PMC3847710. 197. Li, X.; Zhao, L.; Fan, Y.; Jia, Y.; Sun, L.; Ma, S. et al. Occurrence of mycotoxins in feed ingredients and complete feeds obtained from the Beijing region of China. J. Anim. Sci. Biotechnol. 2014, 5, 37. 10.1186/2049-1891-5-37 [PMC free article] [PubMed] [CrossRef] [Google Scholar] 198. Indresh, H. C.; Umakantha, B. Effects of ochratoxin and T-2 toxin combination on performance, biochemical and immune status of commercial broilers. Veterinary World 2013, EISSN: 2231-0916 199. Xue, H. L.; Bi, Y.; Wei, J. M.; Tang, Y. M.; Zhao, Y.; & Wang, Y. New method for the simultaneous analysis of types a and B trichothecenes by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry in potato tubers inoculated with Fusarium sulphureum. Journal of Agricultural and Food Chemistry 2013, 61, 9333–9338. 200. Wangikar, P.; Sinha, N.; Dwivedi, P.K.; Sharma, A. K.. Teratogenic effects of ochratoxin A and aflatoxin B1 alone and in combination on post-implantation rat embryos in culture. Turkish-German Gynecol Assoc. 2007, 8(4) 201. Javed, T.; Bunte, RM.; Dombrink-Kurtzman, MA.; Richard, JL.; Bennett, GA.; Côté, LM.; Buck, WB. Comparative pathologic changes in broiler chicks on feed amended with Fusarium proliferatum culture material or purified fumonisin B1 and moniliformin. Mycopathologia. 2005, 159(4), 553-64. doi: 10.1007/s11046-005-4518-9. PMID: 15983742. 202. Luongo, D.; Severino, L.; Bergamo, P.; De Luna, R.; Lucisano, A.; Rossi, M. Interactive effects of fumonisin B1 and alpha-zearalenol on proliferation and cytokine expression in Jurkat T cells. Toxicol In Vitro. 2006, 20(8):1403-10. doi: 10.1016/j.tiv.2006.06.006. Epub 2006 Jun 30. PMID: 16899350. 203. Szabó, A.; Szabó-Fodor, J.; Fébel, H.; Romvári, R.; Kovács, M. Individual and combined haematotoxic effects of fumonisin B(1) and T-2 mycotoxins in rabbits. Food Chem Toxicol. 2014, 72, 257-64. doi: 10.1016/j.fct.2014.07.025. Epub 2014 Aug 1. PMID: 25092395. 204. Wan, L.Y.; Turner, P.C.; El-Nezami, H. Individual and combined cytotoxic effects of Fusarium toxins (deoxynivalenol, nivalenol, zearalenone and fumonisins B1) on swine jejunal epithelial cells. Food Chem Toxicol. 2013, 57, 276-83. doi: 10.1016/j.fct.2013.03.034. Epub 2013 Apr 4. PMID: 23562706. 205. Kouadio, J.H.; Dano, S.D.; Moukha, S.; Mobio, T.A.; Creppy, E.E. Effects of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 cells. Toxicon 2007, 49(3), 306-17. doi: 10.1016/j.toxicon.2006.09.029. Epub 2006 Oct 11. PMID: 17109910. 206. Pleadin, J.; Frece, J.; Markov, K. Mycotoxins in food and feed. Adv Food Nutr Res. 2019; 89:297-345. doi: 10.1016/bs.afnr.2019.02.007. Epub 2019 Mar 6. PMID: 31351529.