Mycotoxins

C H A P T E R

39 Mycotoxins Wanda M. Haschek1, Kenneth A. Voss2 1

2

University of Illinois, Urbana, IL, USA, USDA Agricultural Research Service, Athens, GA, USA

O U T L I N E 1. Introduction

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2. Aflatoxins 2.1. Source/Occurrence 2.2. Toxicology 2.3. Manifestations of Toxicity in Animals 2.4. Human Risk and Disease 2.5. Diagnosis, Treatment, and Control

1191 1191 1195

3. Ochratoxins 3.1. Source/Occurrence 3.2. Toxicology 3.3. Manifestations of Toxicity in Animals 3.4. Human Risk and Disease 3.5. Diagnosis, Treatment, and Prevention

1203 1203 1203 1205 1207 1208

4. Trichothecene Mycotoxins 4.1. Sources/Occurrence 4.2. Toxicology 4.3. Manifestations of Toxicity in Animals 4.4. Human Risk and Disease 4.5. Diagnosis, Treatment, and Prevention

1208 1208 1209 1212 1220 1221

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1. INTRODUCTION Many species of fungi colonize food crops such as rice, corn (maize), wheat, barley, oats, peanuts, cottonseed, and soybeans, all of which are the basic ingredients of many human and animal foods, including livestock and companion and laboratory animal diets. Under Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Third Edition. http://dx.doi.org/10.1016/B978-0-12-415759-0.00039-X

5. Zearalenone 5.1. Source/Occurrence 5.2. Toxicology 5.3. Manifestation of Toxicity in Animals 5.4. Human Risk and Disease 5.5. Diagnosis, Treatment, and Prevention

1222 1222 1223 1225 1227 1228

6. Fumonisins 6.1. Source/Occurrence 6.2. Toxicology 6.3. Manifestations of Toxicity in Animals 6.4. Human Risk and Disease 6.5. Diagnosis, Treatment, and Prevention

1228 1228 1229 1233 1237 1239

7. Ergot Alkaloids 7.1. Source/Occurrence 7.2. Toxicology 7.3. Manifestations of Toxicity in Animals 7.4. Human Risk and Disease 7.5. Diagnosis, Treatment and Prevention

1239 1239 1241 1244 1247 1248

8. Summary/Conclusion

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Suggested Reading

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certain conditions, fungi produce mycotoxins that can cause adverse effects in other living organisms following exposure. Increasingly, mycotoxins produced by endophytic fungi (an endophyte is an endosymbiont, often a fungus, that lives within a plant for at least part of its life without causing apparent disease) are being shown to be responsible for the toxicity of

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Copyright Ó 2013 Elsevier Inc. All rights reserved.

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39. MYCOTOXINS

poisonous plants. For example, swainsonine found in both Astragalus and Oxytropis spp. is produced by the plant-associated endophyte Undifilum oxytropis (see Selected Poisonous Plants Affecting Animal and Human Health, Chapter 40). Mycotoxins are secondary fungal metabolites (i.e., metabolites not essential to the normal growth and reproduction of the fungus) that cause biochemical, physiologic, and/or pathologic changes in other species, including animals, plants, and other microbes. These effects can range from poor production parameters to death. The word mycotoxin is derived from “myco,” meaning mold, and “toxin,” a poison produced by a living organism. A great number of fungal metabolites have been designated as mycotoxins; however, only a small number are known to have significant animal/human health and economic significance. Mycotoxicosis is the term used to denote a syndrome resulting from poisoning of a biological system by a mycotoxin. Conditions that predispose to mycotoxin production by fungi include appropriate moisture (humidity), temperature, aeration, and substrate type and availability. Temperature and moisture variations affect the growth rate of fungi and also the types and amounts of toxins produced. Individual fungi often produce several different mycotoxins so that combinations of mycotoxins are frequently present, with the possibility of interactive effects (not discussed further in this chapter). Fungi are aerobic organisms, but significant differences in oxygen requirements exist among different species. Fungal interactions with plants are complex: fungi can be saprophytes (fungi that obtain nutrients from dead organic matter) or live as endophytes in the host plant. In either case, environmental stress, insect damage, and plant disease predispose to colonization, growth, and toxin production. In the field, fungi invade both developing and mature seed grains on the plant, and the optimal moisture content for growth is 22–25%. Storage fungi invade grain after harvest while it is in storage; the optimal moisture for growth is 13–18%. Advanced decay fungi (saprophytes) typically require moisture of 22–25% but rarely develop and grow on seed grain in the field. Generally, fungi grow readily between 20
C and 30
C, but optimal temperature ranges can be from below 0
C to above 60
C.

Mycotoxicoses occur worldwide and have been recognized for centuries – for example, St Anthony’s fire (ergot) in the Middle Ages. Many factors contribute to the occurrence of mycotoxicoses in humans, livestock, companion animals, and wildlife. For example, modern harvesting methods in which corn is handled at higher moisture concentrations, combined with damage caused by harvesting machinery, increases the number of kernels in which fungi can initiate growth. Also, feeding ground diets prevents food-producing animals from sorting out and avoiding damaged kernels. A partial listing of mycotoxins is provided in Table 39.1. Our knowledge of many mycotoxins is extremely limited. In many instances, very limited surveys of the frequencies of occurrence have been done. In other instances, the compounds have been investigated in some detail but surveys do not indicate sufficiently frequent occurrence in the United States or other countries for these toxins to be considered of major concern. For example, T-2 toxin and diacetoxyscirpenol are rarely encountered in North America, where T-2 toxin causes only occasional outbreaks of toxicosis in the midwestern USA and western Canada. In contrast, T-2 toxin produced by Fusarium sporotrichioides and F. poae on overwintered grain was likely responsible for severe outbreaks of mycotoxicosis in humans and animals in the Soviet Union. Similarly, the nephrotoxic mycotoxin ochratoxin A is not a major problem in North America, causing only occasional problems in poultry and swine, while it is responsible for widespread outbreaks of toxicosis in swine in Denmark. Four groups of mycotoxins account for at least 95% of the confirmed diagnoses of mycotoxicoses in the midwestern United States. These are fumonisins, deoxynivalenol (DON), zearalenone, and aflatoxins. Other common mycotoxins occurring worldwide are ochratoxin A and trichothecenes other than DON, such as T-2 toxin. Another group of mycotoxins, the ergot alkaloids, causes significant losses in livestock production in the USA, as well as Australia, Argentina, and New Zealand. The primary fungal species producing these toxins are listed in Table 39.2. New mycotoxins continue to be identified. This includes reclassification of previously identified toxins as mycotoxins, e.g., swainsonine, the

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1. INTRODUCTION

TABLE 39.1

A Partial Listing of Mycotoxins

Aflatoxins and structurally related mycotoxins

Zearalenone and zearalenol

Aflatoxins B1, B2, G1, G2, M1, M2, Q1, and aflatoxicol

Moniliformin

Sterigmatocystins

Butenolide

Versicolorins

Fusaric acid

Aspertoxins

Fusarin AeD

Autocystins

Beauvericin

Sterigmatin

Fusaproliferin

Bipolarin

Gliotoxin

Averufarin

Enniatins

Curvularin

Luteoskyrin

Alternariol

Ochratoxin series, especially ochratoxin A

Atranones AeG

Patulin

Citrinin

Penicillic acid

Citreoviridin

Phomopsins

Cyclopiazonic acid

Pyrrocidines

Cyclosporine

Rubratoxins A and B

Diplonene

Sporodesmin

Ergot alkaloids

Tremorgens

Ergotamine

Aflatrem

Ergonovine

Penitrem A

Ergovaline

Roquefortine

Fumonisins

Paspaline

Fusarium toxins

Paspalanine

Trichothecenes

Paspalitrems A and B

T-2 and HT-2 toxins

Verruculogen

Diacetoxyscirpenol (DAS)

Fumitremorgen

Deoxynivalenol (DON)

Fumigaclavine

Verrucarins

Slaframine

Roridins

Swainsonine

Satratoxins

Tenuazonic acid

Table modified from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table I, p. 646, with permission.

cause of locoism, and discovery of new entities such as diplonene, a neurotoxin, produced by the ear-rot fungus Stenocarpella (previously Diplodia) maydis, and responsible for diplodiosis.

This is a neurologic disease of cattle and sheep, and considered one of the most frequently diagnosed mycotoxicosis in ruminants. While the fungus can be found worldwide, diplodiosis

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39. MYCOTOXINS

TABLE 39.2 The Major Mycotoxins and Fungi Producing Them Fungal species

Mycotoxins produced

Aspergillus parasiticus

Aflatoxins B1, B2, G1, G2

A. flavus

Aflatoxins B1, B2

Fusarium sporotrichioides

T-2 toxin

F. graminearum (roseum)

Deoxynivalenol (DON), zearalenone

F. verticillioides, F. proliferatum

Fumonisins

Penicillium verrucosum, A. ochracoeus

Ochratoxin A

Claviceps spp., Neotyphodium coenophialum

Ergot alkaloids

Table modified from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table II, p. 647, with permission.

is most common in South Africa, where cattle and sheep graze the cornfields in winter following harvest. It has also been reported from several countries in South America. In addition to the neurologic syndrome, characterized by ataxia, paresis, and paralysis, the toxin is responsible for stillbirths and neonatal losses. Interpretation of the significance of mycotoxin residues in animal diets is straightforward when extremely high concentrations are present. However, when low concentrations are present in foods, the interpretation of the toxicologic significance of mycotoxin residues can be more difficult. This is because there are differences between mycotoxicoses induced in the laboratory and field cases of mycotoxin poisoning that preclude direct extrapolation of the experimental mycotoxicoses to the situation in the field. In the field, the identified mycotoxin may be consumed along with other related or unrelated fungal metabolites, which may include as yet unidentified mycotoxins. Fungal damaged, stressed grains may be of lower nutrient value and altered palatability. The toxin(s) is (are) unevenly distributed in the diet and the concentration(s) is (are) generally so variable that multiple samples are required to estimate the concentration(s) present. If sufficient moisture and appropriate temperatures occur during transit, additional fungal growth and toxin production may occur before

analysis, potentially confounding diagnostic efforts. The toxin may be bound to, or otherwise associated with, grain or food matrix components in a manner precluding detection or accurate quantification (hidden or masked mycotoxins). Finally, stressors, such as infectious agents, reproduction, lactation, crowding, and temperature variation, overlap and interact with effects of mycotoxins. During experimental mycotoxin administration a single purified toxin is usually administered, the diet is generally balanced and contains undamaged grains, the toxin is evenly mixed in the diet, the toxin concentration is known, the sample is presented to the laboratory without additional fungal growth or toxin production occurring, and a controlled, high-quality environment is provided for the experimental animals. However, interpretation of experimental work presented in the mycotoxin literature can be difficult because of the nature of the toxin administered, the vehicle used, the route and regimen of administration, the numbers and types of animal (species, sex, age, disease status) used, the endpoints examined, the existence of data gaps, and the possibility of multiple plausible interpretations of available data. Although purified toxin is the preferred form of toxin for most experimental studies, limitations on the amount of toxin available (especially when working with larger species) often requires the use of naturally contaminated feed, culture material (grain that has been inoculated with a known toxin-producing fungal strain), or semi-purified toxin. Culture materials and naturally contaminated feed may contain other known or unknown toxins and, as previously mentioned, determination of the concentration of the toxin of interest may be confounded by binding of the toxin to food components. Culture material may also contain other known or unknown toxins, and may have low palatability, which can reduce food intake, especially when it comprises a significant portion of the diet. When food intake is reduced, due either to unpalatability or to toxic effects in the exposed animal, nutritional deficiencies may result (see Nutritional Toxicologic Pathology, Chapter 36). Mycotoxicoses may manifest as acute/subacute, subchronic, or chronic disease. Alternatively, the effects remain subclinical, but there may be growth suppression, decreased weight

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2. AFLATOXINS

gain, and increased susceptibility to nutritional disorders or infection due to immunosuppression. Mycotoxins may be carcinogenic, mutagenic, or teratogenic. As can be seen in Table 39.3, mycotoxins can affect virtually all organ systems and all species; however, each mycotoxin group has limited toxicological targets such that their syndromes are highly distinctive although not pathognomonic. Conversely, there is no syndrome consistent with exposure to mycotoxins as an overall group. The manifestation of toxicity depends on the specific mycotoxin, including exposure dose and time; the species exposed, including age and physiological status; and other factors such as exposure to multiple mycotoxins (not covered in this chapter) and the presence of hidden (“masked”) mycotoxins, i.e., mycotoxins that are not detectable by standard analysis but that can contribute to the exposure. Mycotoxins continue to attract worldwide attention because of their perceived impact on human health, the economic losses accruing from condemned foods and decreased animal productivity, the costs of quality control and monitoring, and the serious impact of mycotoxin contamination on internationally traded commodities. Although pre-harvest control of mycotoxin production is difficult, much effort has been expended to develop resistant crop strains by both breeding and direct genetic modification. Biocontrol technology, in which a non-toxigenic organism competes with a toxigenic fungus for a specific ecological niche (competitive exclusion technology) or otherwise inhibits fungal growth, is being developed. Post-harvest control of mycotoxin production is aimed primarily at effective drying and storage regimens. Approaches utilized to limit exposure, bioavailability, and toxicity of mycotoxins in foodstuffs for animals include the identification and segregation of contaminated material, chemical sorbents used as sequestering agents, and chemical destruction (detoxification). Alternative methods of control are actively being studied because all approaches available have costs and limitations. Strategies for removal of mycotoxins from food materials also need to be developed. Despite the best efforts of the agricultural community, mycotoxins will continue to be present in a wide range of foods. In fact, mycotoxins may actually increase in the food supply

as climate change becomes more severe and as the consumer tries to avoid genetically modified (GM) foods. Genetic modification generally helps the plant to be more resistant to fungal invasion. Therefore, mycotoxins will continue to be a threat to human and animal health and food security worldwide (see Environmental Toxicologic Pathology and Human Health, Chapter 34; Food and Toxicologic Pathology: An Overview, Chapter 35). This chapter addresses the most important mycotoxins that cause significant disease in animals and/or humans. Table 39.4 summarizes the exposure sources, species affected, manifestations, and mechanisms.

2. AFLATOXINS 2.1. Source/Occurrence Aflatoxins are a group of carcinogenic furanocoumarins mainly produced by Aspergillus flavus and Aspergillus parasiticus. These saprophytic fungi are distributed worldwide and are common in the southeastern United States, southern Asia, and Africa, where warm subtropical climates are conducive to fungal growth. These fungi reside in the soil, but under favorable conditions invade the host plants by various routes. Heat, drought, and insect damage to the plants are additional factors favoring infection and aflatoxin production in the field. Fungal growth and aflatoxin production may also begin or, if infection has been established in the feed, continue after harvest in grain that has not been properly dried or is otherwise improperly stored. Aflatoxins can be produced under conditions of 85% relative humidity (e.g., corn moisture content of 15–28%) and temperatures over 25
C that persist for over 48 hours. Under these or other improper conditions, aflatoxin concentrations can become extreme; therefore, proper grain drying and storage are of great importance to minimize hazards to animals and associated economic losses. Corn, peanuts, cottonseed, and tree nuts are common aflatoxin sources; however, these mycotoxins also may be found in other foodstuffs such as wheat, rice, copra, figs, some spices, eggs, and milk. Meats and poultry are not significant sources of aflatoxin; however, aflatoxin M1 (a metabolite of aflatoxin B1) and other residues

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39. MYCOTOXINS

TABLE 39.3

Mycotoxins Classified as to Target Organ Toxicity

Specific agent(s)

Species affected

Time of onset

Usual duration (if survive)

I. MYCOTOXINS THAT CAUSE NEUROTOXICITY

A. Toxicants associated with central nervous system stimulation or seizures Tremorgenic mycotoxins (e.g., penitrem, roquefortine, lolitrem B, paspalitrems)

All species, especially dogs, cattle, sheep, horses

Minutes to days

One day to weeks; often lethal in dogs

B. Toxicants with Mixed Effects on the CNS Ergot alkaloids

Humans

Minutes to days

Days; sometimes lethal

Fumonisins

Horses

Days to months

Permanent damage likely in survivors; often lethal

Diplonene

Cattle, sheep

2e5 days

Few days, reversible

Cattle, horses, sheep

Hours

Up to 3 days; rarely lethal

C. Muscarinic agonists Slaframine

D. Mycotoxins that cause paralysis (may eventually include respiratory paralysis) Lolitrem B

Sheep, cattle, sometimes horses

Chronic

Chronic; rarely lethal

Citreoviridin

Cattle

(Importance in the field is not well established

Patulin

Cattle

(Toxicosis is very rare)

II. MYCOTOXINS THAT CAUSE CARDIOTOXICITY

Citreoviridin (rare) Moniliformin

Poultry

Fumonisin

Pigs, horses

Fusaric acid

Rabbits, rats, cats, dogs, humans

Days

Acute form lethal

Days to weeks

Days to permanent damage; often lethal in poultry

Days to weeks

Days to weeks to permanent damage in rodents

III. NEPHROTOXIC MYCOTOXINS

Ochratoxins

Swine, poultry, humans

Citrinin (may potentiate effect of ochratoxins)

Swine

Fumonisins

Rabbits, lambs, calves, rats, and mice (E)

Cyclosporine

Humans (immunosuppressive use) (Continued)

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2. AFLATOXINS

TABLE 39.3

Mycotoxins Classified as to Target Organ Toxicitydcont’d

Specific agent(s)

Species affected

Time of onset

Usual duration (if survive)

IV. MYCOTOXINS THAT AFFECT THE LIVER

Aflatoxins

Most species, humans; trout, ducklings, and young poultry are highly susceptible

Days to chronic

Weeks to permanent damage, potentially lethal

Sterigmatocystin

Most species

Weeks to chronic

Weeks to months; toxicoses very rare

Rubratoxins A and B

Chickens; possibly cattle and swine

Days to chronic

Unknown; toxicoses rare

Sporodesmin

Cattle, especially sheep

Chronic

Chronic; primarily Australia, New Zealand

Penicillic acid

Swine

Chronic

Chronic; toxicoses very rare

Fumonisins

All species

Days

Weeks; may be lethal in horses

Cyclopiazonic acid (rare) V. MYCOTOXINS THAT AFFECT THE LUNGS

3-Substituted furans, e.g., ipomeanol

Ruminants

Hours to days

Days; often lethal

Fumonisins

Swine

Days

Usually lethal

Stachybotrys toxins, e.g., satratoxins

Humans, horses, cattle, pigs, sheep, poultry (when inhaled)

VI. MYCOTOXINS THAT CAUSE IMMUNOSUPPRESSION

Aflatoxins

All species

Trichothecenes

All species

Fumonisins

Pigs, rodents (E)

Ochratoxin A

Pigs (E)

VII. MYCOTOXINS THAT INDUCE CANCER

Aflatoxins

Most species, trout, humans (liver)

Ochratoxin A

Mice, rats (kidney and urinary tract) (E)

Fumonisins

Rat (kidney and liver), mouse (liver) (E)

Sterigmatocystin

Rat (liver) (E) (Continued)

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TABLE 39.3

Mycotoxins Classified as to Target Organ Toxicitydcont’d

Specific agent(s)

Species affected

Time of onset

Usual duration (if survive)

VIII. MYCOTOXINS THAT ARE ENDOCRINE DISRUPTORS AND AFFECT REPRODUCTION AND MAMMARY GLAND FUNCTION

A. Mycotoxins that are estrogenic Zearalenone, zearalenol

Swine, cattle, sheep

Days to weeks

Days to weeks, permanent reproductive damage rare

Acute (Claviceps sp.)/ chronic (Fescue)

Chronic

B. Other mycotoxins that affect reproduction Ergot alkaloids

Cattle, horses

C. Mycotoxins that affect the mammary gland or lactation Ergot alkaloids

Swine, cattle, horses

Days to months

Days to months; rarely lethal

Zearalenone

Swine, cattle

Days to weeks

Days to weeks (not lethal)

IX. MYCOTOXINS THAT AFFECT THE GASTROINTESTINAL TRACT

Deoxynivalenol (DON)

Swine, cattle, dogs, and poultry

Hours to days

Days; unlikely to be lethal

T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), and other trichothecenes

Cattle, swine, small animals, poultry, humans

Hours to chronic

Days; potentially lethal

X. MYCOTOXINS THAT MAY AFFECT THE SKIN

T-2 toxin (dermal exposure)

All species

XI. MYCOTOXINS THAT AFFECT PERIPHERAL CIRCULATION (MAY CAUSE SLOUGHING)

Ergot alkaloid (gangrenous ergotism)

Cattle, sheep

Days to weeks

Weeks; potentially lethal

Ergot alkaloids in tall fescue (fescue foot)

Cattle

Weeks to months

Weeks to months

E, experimental. Table modified from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table III, pp. 648–650, with permission.

can be found in the muscle of exposed animals. The presence of aflatoxin M1 in milk is of concern as a potential exposure source to infants and children consuming milk, cheese, and other dairy products. Aflatoxins were first identified as the causative agent of an acute and fatal disease in turkeys, called Turkey X disease. This outbreak occurred in England in 1960, and affected over 100 000 birds that were fed moldy groundnut (peanut) meal from Brazil. Since then, the

aflatoxins, especially aflatoxin B1, have been the most thoroughly studied mycotoxins, both in the laboratory and as the subject of epidemiology studies of mycotoxins and human disease. Although classically regarded as hepatotoxins and hepatocarcinogens, aflatoxins also exert adverse effects on other tissues, most notably the kidneys and the hematopoietic and immune systems. Exposure has more recently been associated with poor growth in neonates and children.

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2. AFLATOXINS

2.2. Toxicology Toxin At least 13 aflatoxins have been identified. Aflatoxins B1, B2, G1, and G2 are the most common, with aflatoxin B1 being the most important as it is a potent toxin, mutagen, and carcinogen. It is also the most studied (Figure 39.1). The designations B and G refer to visualization of these compounds in peanuts, corn, and other commodities by the bluish (B) or greenish (G) fluorescence that they emit under ultraviolet light. Aflatoxins G1 and G2 are produced only by Aspergillus parasiticus. Aflatoxins B2, G1, and G2 are less potent than aflatoxin B1, and the metabolites M1 and M2 are considerably less potent than their precursors. Species susceptibility Aflatoxin B1 is toxic to some degree in all species tested to date, although significant differences in sensitivity occur. Among agriculturally important mammals, pigs are generally more sensitive than cattle, which in turn are more sensitive than sheep. Ducklings and turkey poults are more sensitive than quail or chicks. Young animals are, as a rule, more susceptible than adults. Adult mice are less sensitive than neonates as well as adults of other laboratory species including rats, guinea pigs, and rabbits. Aflatoxin B1 induces hepatocellular carcinoma in rats. Grown mice are resistant; however, lung tumors have been induced in mice following aflatoxin B1 administration. Trout have been shown to be very sensitive to the hepatocarcinogenic effects of aflatoxin and have served as a model for large-scale carcinogenicity studies (see Alternative Animal Models, Chapter 14). Biodistribution, Metabolism, and Excretion Aflatoxins B1, G1, and others are procarcinogens that are subjected to both phase 1 and phase 2 metabolism in the liver and other tissues. Cytochromes P450 (CYP) are responsible for their phase 1 metabolism in mammals, with metabolism in humans being mediated by several, including CYP1A2, CYP3A5, and CYP3A4. Phase 1 metabolism leads to formation of less toxic molecules as well as more reactive, electrophilic species that readily react with cellular macromolecules. CYP1A2-catalyzed conversion of aflatoxin B1 to aflatoxin M1, estimated to be 10 times less

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potent a carcinogen than aflatoxin B1, is an example of the former. Aflatoxins P1 and Q1 are further examples of less toxic oxidative metabolites (see Pharmacokinetics and Toxicokinetics, Chapter 2). Aflatoxin B1 is bioactivated by its conversion to exo aflatoxin B1-8,9-epoxide which, like other bioactive epoxides, undergoes further reactions, including phase 2 glutathione-S-transferase mediated conjugation to glutathione (with subsequent urinary excretion as aflatoxin-mercapturic acid), conversion to dihydrodiols or dialdehydes, and covalent binding to macromolecules. Covalent binding of aflatoxin epoxide to DNA occurs, resulting in adduct formation. The N7 sites of guanine nucleosides are particularly susceptible. The aflatoxin–N7 guanine adduct, which is a depurination product formed during DNA repair, can be found in blood and urine and has proven to be an extremely useful mechanism of action-relevant biomarker of recent exposure in epidemiological and other investigations. Aflatoxin–albumin adducts found in serum and aflatoxin-mercapturic acids in urine are additional metabolic products that are also useful biomarkers of exposure. Differences among species and gender in susceptibility to aflatoxicosis are explained in part by differences in metabolism rates and the amounts and types of phase 1 and phase 2 metabolites formed. For example, the relative insensitivity of adult mice to hepatocarcinogenicity can be attributed to their significantly higher levels of glutathione-S-transferase activity compared to rats and other more sensitive species. However, cytosolic phase 2 metabolism of aflatoxin is significantly less efficient in humans than in mice. The importance of phase 2 metabolism for detoxification is illustrated further by the protective effects observed in animals treated with agents such as Oltipraz (a dithiolethione) that induce glutathione-S-transferase activity. Mechanism of Action Aflatoxins are acutely hepatotoxic. The underlying mechanism is widespread and non-specific interactions between aflatoxins or their activated metabolites and various cell proteins, leading to disruption of basic metabolic processes and protein synthesis that, in turn, can cause cell death. Aflatoxins also are genotoxic. The correlation between aflatoxin exposure and the appearance

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TABLE 39.4

Major Mycotoxins, Sources, Species, Manifestations and Mechanisms Primary pathology or manifestation

Mycotoxin Aflatoxins, specially B1

Fumonisins, especially B1

Main fungal producer(s)

Exposure source (ingestion)

Species affected

Acute

Chronic

Mechanism(s)

Aspergillus spp., Corn, peanuts, e.g., A. flavus, cottonseed, treenuts, A. paraciticus contaminated pet food

Cattle, pigs, dogs, horses, birds, fish, humans, all susceptible

Liver: zonal necrosis, lipidosis, biliary hyperplasia

Liver: megalocytosis, biliary fibrosis, cirrhosis, cancer

Acute aflatoxicosis

Chronic aflatoxicosis

Electrophile, cytochrome P450 bioactivation, DNA and macromolecule adducts

Fusarium verticillioides, F. proliferatum

Horses, pigs, all laboratory animals (mammals) susceptible

Liver: apoptosis, necrosis

Liver: fibrosis (pigs, horses); neoplasia (rodents)

Pigs

Lung edema due to cardiotoxicity

Liver: fibrosis

Equids: horses, donkeys

Central nervous system: vasogenic edema, leukoencephalomalacia (ELEM)

Liver: fibrosis

Experimental: rat, rabbit, lamb, calf

Kidney: tubular epithelial apoptosis

Kidney: cancer (rats)

Corn

Alteration of sphingolipid metabolism and function

Penicillium ochraceus and Aspergillus verrucosum

Grain, grapes, coffee, pork products

All species susceptible, pigs most commonly exposed

Kidney: tubular degeneration/necrosis, proximal tubules

Kidney: interstitial fibrosis (pig, ? human); cancer (rat, ? human); BEN (? human)

Cytochrome P450 bioactivation; inhibit mitosis; proapoptotic

Deoxynivalenol

Fusarium graminearum, F. culmorum

Grain, especially wheat and barley

Pig most sensitive, all susceptible

No specific pathology. High dose, emesis; lower dose, feed refusal

Reduced feed intake and weight gain, immune dysregulation (all species likely); IgA glomerulonephritis (mouse, ? human)

Hormonal and cytokine dependent dysregulation of appetence/satiety involved

T-2 toxin

Fusarium sporotrichioides, F. poae

Grain, especially barley and wheat

Pig and cat most sensitive, all susceptible

Systemic: hematopoetic, lymphoid, gastrointestinal e apoptosis, necrosis. Local: skin, oral mucosa e irritation. ATA (human)

Infection due to immune suppression

Inhibit protein synthesis and mitochondrial function, activate MAPKs, alter neurotransmitters

39. MYCOTOXINS

Ochratoxins, especially A

Straw, hay (ingestion, contact)

Horse, human (stachybotryotoxicosis)

Systemic: hematopoetic, lymphoid, gastrointestinal e apoptosis, necrosis. Local: skin, oral mucosa e irritation

Stachybotrys chartarum

Water damaged buildings (inhalation)

Human; experimental: monkey, laboratory animal

Respiratory, central nervous system. “Sick building syndrome”

Zearalenone

Fusarium graminearum

Corn, wheat

Pig most sensitive, all presumed susceptible

Reproductive and mammary gland: estrogenic effects

Ergot alkaloids

Claviceps spp.

Grain, especially rye

All species susceptible

Ergotism. Gangrene (vascular smooth muscle hyperplasia/ hypertrophy), hyperthermia, reproductive (abortion, agalactia), neurologic and enteric syndromes

Ergot alkaloids, mainly ergopeptine alkaloids

Neotyphodium coenophialum

Fescue grass

Cattle and horses mainly

Assume similar to T-2 toxin

Endocrine disruption: acts on estrogen receptors a and b Similar to acute if lower exposure dose

Endocrine disruption due to neurohormonal effects: agonist/ antagonist action on adrenergic, dopaminergic and serotonergic receptors resulting in vasoconstriction and uterine smooth muscle contraction

Fescue toxicosis. Cattle: gangrene (fescue foot), hyperthermia with temperature stress. Horses: reproductive (delayed parturition, agalactia). Vascular smooth muscle hyperplasia/hypertrophy in bovine peripheral tissues and equine placenta

Endocrine disruption and vasoconstriction as above; D2 dopaminergic agonism results in decreased prolactin leading to agalactia

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Stachybotrys, Myrothecium spp.

Macrocyclic trichothecenes

BEN, Balkan endemic nephropathy; ATA, alimentary toxic aleukia.

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adduct and serum hepatitis B surface antigen than in men having only the viral surface antigen. Nutritional considerations and oxidative damage to DNA and other macromolecules caused by aflatoxin-induced lipid peroxidation also may play an important mechanistic role.

2.3. Manifestations of Toxicity in Animals

FIGURE 39.1

Chemical structure of aflatoxin B1.

of N7 guanine–aflatoxin adducts in urine has been repeatedly demonstrated. As a consequence of DNA adduction, mutations occur during DNA repair or replication and, if involving critical genes, they can significantly alter cell functions. One example of this phenomenon that has implications for human health is the high correlation between aflatoxin exposure and a characteristic point mutation found at the third base of codon 249 of the TP53 tumor suppressor gene. This mutation, a transversion of guanine to thymidine (AGG to AGT), is present at relatively high frequency in Chinese and African liver cancer patients. However, some studies suggest that aflatoxin carcinogenesis is independent of TP53 mutations. Though classified as a human carcinogen, there is a body of evidence that aflatoxin requires the presence of other factors to induce liver cancer. In this regard, the role of hepatitis B as a cofactor for aflatoxin has gained much attention, as infection with hepatitis B virus is common in areas where aflatoxin exposure is high, such as Africa and Southeast Asia. Some epidemiological studies using hepatitis B surface antigen as a biomarker for viral exposure indicate that carcinogenesis is likely related to an interaction between hepatitis B virus and aflatoxin. For example, results of a cohort study in China indicated that the relative risk of liver cancer was significantly greater among men who were positive for both urinary N7 guanine

Overview The aflatoxin literature is extensive, more so than for any other mycotoxin. A comprehensive treatment of the subject is therefore beyond the scope of this chapter. The reader is referred to the extensive reviews of the toxicity and pathology of aflatoxins, especially the hepatic histopathology, by Kensler and colleagues (see Suggested Reading section) and by others. Acute aflatoxin poisoning occurs less commonly than chronic aflatoxicosis. The principal target organ is the liver, with hepatocellular fatty change, degeneration and necrosis. Loss of liver function results in icterus, decreased synthesis of serum proteins (hypoproteinemia), and coagulopathy, due to decreased synthesis of clotting factors. Coagulopathy can lead to extensive hemorrhaging and anemia. Clinical signs of chronic aflatoxicosis are non-specific, but biochemical evidence of hepatocyte and biliary damage can be obtained from serum. Chronic intoxication is associated with decreased weight gain or weight loss, decreased food consumption and conversion, and decreased reproductive performance, including abortion. Laying hens exhibit reduced egg production, and milk production of cows declines. Affected animals have increased susceptibility to infection, presumably due to immunosuppression: aflatoxins have an adverse effect on cell-mediated immunity, principally by affecting the reticuloendothelial system, macrophages, and T cells. Although affecting other tissues, aflatoxins usually are thought of in terms of hepatotoxicity and hepatocarcinogenicity. There is a reasonable degree of similarity in the type of liver lesions seen in different species, both on the gross and microscopic levels and in accompanying clinical pathology changes. Some variability exists, for example, the distribution of hepatic necrosis tends to be centrilobular in some species (guinea pig, dog, pig, cattle) and more periportal in

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FIGURE 39.2 Liver from a dog fed aflatoxin contaminated pet food. The liver is swollen and yellow because of hepatocellular lipidosis. The gall bladder is distended. Photograph courtesy of Dr B. Summers, Cornell University. Figure reproduced from Fundamentals of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), Fig. 9.11A, p. 215, with permission.

others (rat, poultry, cat). Qualitatively, the lesions caused by the various aflatoxins appear similar, although there is a difference in potency: aflatoxin B1 is more potent than aflatoxin G1 and far more so than aflatoxin M1. Grossly, the liver has been variably described as enlarged, swollen, or fatty; it tends to be pale with gray to yellow or orange discoloration (Figure 39.2). Congestion or petechial hemorrhages are sometimes evident. The texture is variable, and may be firm, fibrous, friable, or fatty, particularly in chickens and dogs. The gall bladder may be enlarged and turgid with mucosal hemorrhage. Splenic or renal enlargement, hydrothorax, hydropericardium, or ascites may also be found. Microscopic lesions caused by acute or subchronic exposure in most species include hepatocyte degeneration, necrosis, hepatocellular vacuolation (fatty change), cellular pleomorphism with variability in cell (anisocytosis) and nuclear (anisonucleosis) size, bile duct or oval cell proliferation (Figure 39.3), and nodular regeneration, which may progress to cirrhosis or cancer. The predominance of individual findings such as bile duct proliferation, distribution of necrotic hepatocytes, degree of lobular architectural

FIGURE 39.3 Chronic aflatoxicosis; liver from a dog fed aflatoxin contaminated pet food. H&E stain. (A) Hepatocytes are pale due to cytoplasmic vacuoles (lipid) except for a focal area of hepatocellular hyperplasia (lower right). Bile duct proliferation is noted in periportal areas (arrows). A centrilobular vein is present in the center of the field. (B) Higher magnification demonstrates bile duct hyperplasia and hepatic lipidosis. Multiple bile ductules (arrows) extend from the portal area. Adjacent hepatocytes contain variably sized cytoplasmic vacuoles (lipid).

disruption, and time course of lesion development varies by species and mode of exposure. Laboratory Animals Rats are sensitive to the acute hepatotoxic effects of aflatoxin, but less so than guinea pigs and more so than mice. Lesions are progressive in rats given a single LD50 dose. Shortly after dosing, there is periportal degeneration and

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necrosis of hepatocytes with biliary and oval cell proliferation. Later, hepatocytes become more pleomorphic with nuclear hyperchromasia and differences in nuclear size and shape. Regeneration becomes more obvious until overt nodular regeneration is present. Fibrosis may not be extensive, and hepatocellular tumors, which morphologically resemble those routinely induced by other rodent hepatocarcinogens, develop in the absence of cirrhosis. Similar non-neoplastic lesions are found in mice, guinea pigs, and rabbits; however, hepatic necrosis in guinea pigs is typically centrilobular and in rabbits is centrilobular to midzonal. Poultry Poultry, particularly ducklings, are sensitive to aflatoxins. As in mammals, biliary hyperplasia is a predominant feature. Hepatocellular degeneration and necrosis occur. Necrotic cells may be scattered throughout the hepatic lobules or, more commonly, are found in the periportal zone. In ducklings, periportal hepatocellular necrosis may be accompanied by the development of lesions described in the literature as “lakes of fat,” representing intracellular lipid accumulation. Biliary hyperplasia and nodular regeneration become pronounced, and fibrosis progresses to cirrhosis. Livestock Aflatoxicosis is a problem in livestock, most notably swine and cattle. Young pigs are especially sensitive to acute exposures. Gross lesions include hepatic enlargement, congestion, yellow discoloration, and friability; petechiae or more generalized hemorrhage; and edema and ecchymotic or petechial hemorrhages of the gall bladder. Microscopic findings depend upon the dose and duration of exposure but are typical of aflatoxicosis, with hepatocellular degeneration and necrosis in the centrilobular zone. Cattle are more resistant than pigs, but the typical lesions of aflatoxicosis, as described above, can be found following exposure. Fibrosis and bile duct proliferation may be extensive and found together with fibrotic veno-occlusion of the central veins. Sheep are resistant to aflatoxin. Other Species Other species, including non-human primates, have shown varying degrees of sensitivity to

aflatoxin and develop lesions of the type described above, particularly hepatocellular degeneration and necrosis, and biliary proliferation that progress to nodular cirrhosis. Dogs are quite sensitive, presumably in part due to low hepatocellular glutathione levels as compared to other species. Outbreaks of toxicoses associated with commercial pet food made with corn contaminated by high levels of aflatoxins are reported periodically in the USA. Acute exposure results in jaundice and liver and gall bladder lesions. Grossly, the livers can be enlarged and yellow (see Figure 39.2). Microscopic findings are consistent with those found in other species and include centrilobular hepatocyte degeneration, lipid vacuolation, and biliary hyperplasia (see Figure 39.3). Clinical findings in cases of acute or subacute exposure include elevated liver enzymes, hyperbilirubinemia, hypoproteinemia, hypocholesterolemia, and reduced clotting activity. Aflatoxin toxicity is not confined to mammalian and avian species, but extends to fish, with trout being particularly sensitive. The acute and subchronic effects include hemorrhage and hepatocyte necrosis. Biliary proliferation, regenerative nodules, and hepatocellular carcinoma are common findings after prolonged exposure. Liver Cancer in Laboratory Animals The lesions associated with aflatoxin hepatocarcinogenesis were thoroughly studied and reported in the late 1960s and early 1970s (see Suggested Reading section). These investigations not only brought attention to the carcinogenic potential of aflatoxins, but also established mycotoxins as important environmental toxins. The morphology of hepatic neoplasms induced by aflatoxins is similar to those induced by other well-characterized carcinogens. However, in contrast to tumors induced by some other compounds, aflatoxin-induced hepatomas and hepatocellular carcinomas can arise in livers that are not cirrhotic. The sequence of lesions that occur in rats fed aflatoxin B1 ( 1 ppm in the diet) was described in detail by Newberne and Wogen. In the early stages, there is bile duct proliferation, and foci of altered hepatocytes consisting of intensely stained cells with small nuclei or, alternatively, larger cells with clear to lightly staining cytoplasm. With continued exposure, hyperplastic

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nodules, hepatomas, and overt carcinomas develop. The hyperplastic nodules contain welldemarcated collections of hepatocytes undergoing mitosis or fatty change that compress the surrounding parenchyma. Carcinomas range in appearance from well differentiated, with cells arranged in trabeculae, to poorly differentiated types. In the latter, cells display varying degrees of anaplasia and may be arranged in sheets, cords, cysts, or duct-like structures. The tumors invade the adjacent parenchyma and vasculature, and frequently metastasize to the lung. Cholangiofibromatous lesions and cholangiocarcinoma are rare.

2.4. Human Risk and Disease Acute aflatoxicosis following the ingestion of highly contaminated food has been documented in various locations, particularly in Africa and southeastern Asia. Diagnosis of aflatoxicosis in humans is difficult, as symptoms are not specific for aflatoxin. Clinical findings may include anorexia, diarrhea, malaise, or depression. Death may occur. Hepatobiliary involvement is indicated by jaundice, ascites or tenderness when pressure is applied to the upper abdomen. Histopathological findings consistent with aflatoxin-induced injury, such as fatty infiltration and centrilobular necrosis, have been reported following acute outbreaks of suspected human aflatoxicosis. Severe outbreaks of acute aflatoxicosis have occurred from time to time. In India, over 100 fatalities occurred in 1974 when unseasonable rain and a scarcity of food resulted in consumption of corn that was heavily contaminated with aflatoxin. Kenya has had periodic outbreaks of aflatoxicosis since 1972, including the severe episode of January through July 2004 that involved 317 known cases and claimed the lives of 125 individuals. In one survey, aflatoxin levels of up to 58 ppm were found in corn samples from the outbreak area and concentrations exceeding 1 ppm were found in more than half of corn samples that were associated with illness. While cases were associated mostly with consumption of homegrown corn, aflatoxin contamination in corn sold at local markets was also extensive and contributed to exposure: more than 50% of the market samples exceeded the Kenyan regulatory standard of

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20 ppb. Additional cases of acute aflatoxicosis in Kenya were identified in 2005. Aflatoxin B1 has been classified as a human carcinogen, with liver cancer being the major concern. While there is considerable uncertainty due to methodological limitations, it has been estimated that aflatoxin is involved in up to 155, 000 cases worldwide, corresponding to as much as 28% of all cases of liver cancer per year. The association between aflatoxin and liver cancer has been a focus of intense epidemiologic investigation in the developing world, particularly in China and various sub-Saharan African countries where high liver cancer incidence is found. Significant exposure in these regions has been unequivocally demonstrated using aflatoxin–lysine adducts in serum and aflatoxin B1–N7guanine adducts as biomarkers. However, nutritional and other conditions frequently exist in affected human populations, which confound the situation. Of particular interest is exposure to hepatitis B virus, which is now generally accepted to be a potentially important cofactor. However, epidemiological and other investigations have yielded mixed results: some suggest that aflatoxin acts independently, some correlate liver cancer with hepatitis B exposure, and still others show an interaction between the two. Recent studies in China and Thailand, in which biomarkers were used to assess exposures, indicate a strong likelihood that both aflatoxin B1 and hepatitis B virus are involved and that an interaction between the two contributes to risk. This is supported by the findings of a recent in-depth review and metaanalysis of multiple epidemiological studies on aflatoxin-related liver cancer risk: (1) aflatoxin is by itself a significant risk factor and (2) in regions where aflatoxin exposure is high and chronic hepatitis B virus is prevalent, the two factors work synergistically or “multiplicatively” to increase cancer risk. The study in Thailand showed the strongest correlation occurring in hepatocellular carcinomas occurring without concurrent cirrhosis. There is increasing evidence that other human health risks are associated with aflatoxin exposure, including growth impairment and immune disorders. Stunting of growth is associated with poor health outcomes in later life, including decreased resistance to infection. Both cross-sectional and longitudinal epidemiological studies in West

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Africa revealed significant correlations between growth impairment and serum aflatoxin–albumin adduct levels in young children. Transition to solid food was a critical contributor to exposure, as significantly greater adduct levels were found in fully weaned compared to partially weaned children. Prenatal exposure might also be critical and, in this regard, high levels of aflatoxin– albumin adduct in maternal blood during pregnancy have been associated with weight and height deficits in children during the 4 months following birth. While implicating aflatoxins, the issue is not resolved and any physiological mechanism by which aflatoxin, directly or indirectly (in conjunction with other risk factors), affects growth remains to be elucidated. Secondly, the aflatoxin findings might be serving as a surrogate marker for other conditions that impact growth, such as a vitamin or other nutrient deficiency, gut microflora composition, or parasite burdens. The impact of aflatoxin exposure on the immune system is likewise not clear. Results of animal studies vary by species and experimental design, but have nonetheless shown adverse impacts on components of both cellular and humoral immune responses. Targets include T lymphocyte populations, monocytes and macrophages, and, accordingly, cytokine and antibody production, as well as proliferation and phagocytic functions, were impaired in some experimental models. The number of studies evaluating aflatoxin and human immunity is limited, but the results do suggest that exposure is potentially detrimental. Among the reported effects associated with high levels of serum aflatoxin–albumin adducts in West African populations are reductions in the number of activated T (CD4þCD69þ) cells, including cytotoxic T cells staining positive for perforin or gramzyme, decreased numbers of activated B (CD18þCD69þ) cells, and decreased salivary levels of secretory IgA in children. A preliminary positive correlation between serum aflatoxin–albumin adducts and high tissue burdens of HIV virus has been reported.

2.5. Diagnosis, Treatment, and Control When conditions are favorable for production of aflatoxin, grain elevators often use black light (ultraviolet) to screen commodities. While simple and quick, this test only indicates fungal growth and not toxin presence. All positive findings

must be followed up using a specific test to identify and quantify any aflatoxins actually present. USDA Grain Inspection, Packers, and Stockyards Administration (GIPSA)-approved test kits are available for both qualitative screening for aflatoxins in corn and quantitative determination of aflatoxin concentrations in corn and other commodities. Qualitative methods give a positive or negative result, with the “cut-off” in the United States being 20 ppb, the actionable limit concentration set by US Food and Drug Administration (FDA) for total aflatoxins in food and nuts for human consumption. Action levels for animal feeds vary from 20 (most species) to 300 ppb (beef cattle, non-breeding) depending not only on the species but also on physiologic status. Aflatoxin M1, a hydroxylated metabolite of aflatoxin B1, is readily detected in the milk of exposed dairy cattle, and the actionable limit is < 0.5 ppb aflatoxin for the sale of milk. Diagnosis of aflatoxicosis in a clinical or field setting cannot be based on lesions and clinical pathology findings alone. Confirmation of diagnosis requires direct evidence of aflatoxin exposure, such as the identification of aflatoxin– albumin adducts in serum, the presence of aflatoxin–N7 guanine adducts in the urine, identification of sufficient concentration of aflatoxin(s) in source materials or, preferably, a combination of the above. Treatment of affected animals consists of changing the diet to an aflatoxin-free ration, increasing dietary protein, and supplementing the diet with Vitamin B12, Vitamin K, and selenium. Toxicity can be prevented to some extent by treatment of contaminated foodstuffs. NovaSil7, a hydrated sodium calcium aluminosilicate (HSCAS), binds aflatoxin. When added to aflatoxin-contaminated feed, NovaSil7 and some other clays have been shown to decrease aflatoxin absorption and toxicity in swine and poultry. However, the efficacy of NovaSil7 in preventing residues of aflatoxin M1 in the milk of dairy cows is less than desirable. NovaSil7 has been approved by the FDA only as an anticaking agent at up to 2% of a ration. Ammoniation of corn to reduce its aflatoxin concentration is a last resort only, and ammoniated corn turns brown. Ammoniation has been used extensively to detoxify aflatoxin in cottonseed intended for animal consumption. Ammoniation of corn for animal consumption has been approved by

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some states, but not by the FDA, and interstate shipment of treated corn is illegal. Both NovaSil7 and ammoniation can be used only for animal feed. However, while not approved for humans, NovoSil clay given in capsule form has been tested in a 3-month Phase 2a clinical intervention trial in Ghana. Treatment reduced the levels of aflatoxin–albumin adducts found in the serum compared to the group given placebo, suggesting that NovoSil clay can reduce the bioavailability of aflatoxin found in foods. The introduction of non-aflatoxigenic Aspergillus strains as competitive organisms in the field has been shown to reduce aflatoxin concentrations in cottonseed and peanuts.

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grapes, including wine and raisins. They may also occur in other commodities, such as cottonseed, nuts, and dried beans. Meats, especially kidney or pork products such as sausages, bacon, or ham, also contribute to human exposure. Geographically, ochratoxins are found in regions having temperate climates, with the northern European countries, the Balkans, and Canada being most affected. Optimal conditions for ochratoxin A production are a moisture content of 19–22% and a temperature of 24
C. Ochratoxin concentrations in grains are variable, but can periodically be high enough to cause outbreaks of porcine nephropathy or other animal diseases. Human exposure is particularly high within, but not limited to, the Balkans and northern Europe. Ochratoxin A residues have been found in human serum, plasma, and milk from northern European countries.

3.1. Source/Occurrence Ochratoxins A, B, and C are secondary metabolites of Aspergillus ochraceous, Aspergillus carbonarius, Aspergillus niger, Penicillium verrocusum, and related species. In addition, ochratoxin methyl esters, ethyl esters, and other analogs have been characterized. Ochratoxins A, B, and C contain a phenylalanine moiety attached to a dihydroisocoumarin group via an amide bond (Figure 39.4). Ochratoxin A is the most common and most important from an animal and human health standpoint. It is nephrotoxic to multiple species, and is a potent renal carcinogen in rodents. Humans and animals can be exposed via the diet. The principal source of ochratoxins is cereal grains, including barley, rye, wheat, corn, sorghum, and oats, as well as coffee beans and

3.2. Toxicology Species Susceptibility The toxicities of ochratoxin A and ochratoxin C are approximately equal, although the former is much more commonly encountered. Ochratoxin B is several times less potent. Synergistic or additive effects have been described in animals co-exposed to ochratoxin A and other mycotoxins such as aflatoxin, citrinin, and penicillic acid. Ochratoxins are potentially hazardous to livestock, and toxicity has been demonstrated in swine, horses, ducklings, chickens, turkeys, and dogs. Cattle are relatively resistant due to metabolism of the mycotoxin by ruminal microflora.

FIGURE 39.4 Chemical structure of ochratoxin A and phenylalanine. Ochratoxins compete with phenylalanine for binding sites of enzymes.

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Young animals are in general more sensitive than adults, including calves, which remain so until the rumen becomes fully functional. The kidney is the major target organ in swine and other species, although the liver, immune system, and other organs also may be affected. Ochratoxin A is teratogenic in most species examined, and dose-dependent transfer across the placenta has been demonstrated in rodents. Swine are a notable exception. In contrast to rodents, ochratoxin A does not cross the placenta when given to sows at low levels and thus teratogenic effects do not occur. Biodistribution, Metabolism, and Excretion The pharmacokinetics of ochratoxin A in mammals vary depending on species and dose. In general, about 60% of an orally administered dose is absorbed from the gastrointestinal tract of rats and other monogastric animals. In rats, significant amounts bind to plasma albumin, with maximum serum concentrations occurring within 4 hours of dosing. Binding to serum albumin is also high in cattle, pigs, and humans. The oral t1/2 varies from about 8 hours in rabbits to about 120–230 hours in rats to slightly over 500 hours in monkeys. The t1/2 in pigs is about 90 hours, and the Cpmax (maximum plasma concentration) reportedly ranges from 0.50 mg/ mL or less in mice and monkeys to as high as 87 mg/mL in rats. Biliary excretion and enterohepatic circulation occur, and ochratoxins and their metabolites are excreted in urine and feces. The high level of binding to serum albumin and greater retention rates in pigs leads to the accumulation of residues in tissues, most notably the kidneys, where amounts of up to 100 ppb have been documented. Lower levels can accumulate in the tissues of chickens. Rats also accumulate ochratoxin in the kidney so that its concentration therein is about 40-fold higher than in liver 24 hours after exposure. The anionic transporter mechanisms play a major role in renal accumulation, and absorption of the mycotoxin occurs along the entire length of the renal tubule. Interestingly, ochratoxin A has been shown to downregulate genes coding for some proteins involved in ochratoxin A transport. Other tissues, especially liver, skeletal muscle, and fat, accumulate ochratoxin A to a lesser extent.

Ochratoxin A is metabolized to a less toxic hydrolysis product, ochratoxin a, by carboxypeptidases found in the rumen and intestine; enteric bacteria are the likely source of the enzymes. In addition, hepatic and renal metabolic pathways found in various species, including rat, mouse, rabbit, and monkey, convert ochratoxin A to ochratoxin B, 4-R- and 4-Shydroxyochratoxin A, 10-hydroxyochratoxin A, pentose or hexose mycotoxin conjugates, or other uncharacterized products. Conversion to reactive oxygen species (ROS) is also a possibility (see below). Metabolism varies by species, sex, and strain of animal, and is mediated by cytochrome P450s. Mechanism of Action As a result of their structural similarity to phenylalanine (Figure 39.4), ochratoxins effectively compete with phenylalanine for the binding sites of enzymes that utilize the latter as a substrate. The results of all potential metabolic inhibitions, e.g., the inhibition of phenylalanine hydroxylase, are not known. Ochratoxins do, however, inhibit phenylalanine tRNA synthetase and, as a result, cellular protein synthesis is reduced. Ochratoxins also inhibit mitochondrial respiration, leading to depletion of cellular ATP, disrupted calcium homeostasis, lipid peroxidation, and oxidative damage of macromolecules. Phenylalanine and aspartame (which contains phenylalanine) are antagonistic to ochratoxins, presumably by competing for critical phenylalanine binding sites. The molecular mechanism(s) and mode of action underlying the toxicity and carcinogenicity of ochratoxin A, including the critical issue of whether or not OTA is genotoxic, are controversial. Evidence supporting a genotoxic mode of action comes largely from 32P-postlabeling studies in which putative ochratoxin A– or ochratoxin A metabolite–DNA adducts co-migrate chromatographically with a photochemically generated ochratoxin A–DNA reaction product, putatively a C8-deoxyguanosine–ochratoxin A compound. It has been hypothesized that adducts form via conversion of ochratoxin A by cytochrome P450 to an electrophilic ochratoxin A quinone, by reductive dechlorination to an aryl radical, or by peroxidase and glutathione mediated conversion to a phenoxyl radical. In turn, these reactive metabolites might bind DNA. The detection of

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ochratoxin A-hydroquinone, which is formed by further reduction of ochratoxin A-quinone, in urine and kidney of rats as well as in human urine and blood from the Balkans has been reported. While this is consistent with the hypothesized mechanism, isolation and unequivocal structural confirmation of ochratoxin A or ochratoxin A metabolite adducts from animal or human tissues has not as yet been accomplished. The issue of mutagenicity continues to be unresolved. Ochratoxin A does not elicit a mutagenic response in the Salmonella typhimurium T100-, T102-, and T102-related tester strains but is mutagenic in others, including T98, when the procedure is modified to include metabolic activation by mouse kidney microsomes. Results of other genotoxicity tests, such as sister chromatid exchange and unscheduled DNA synthesis, have also been inconsistent. Non-genotoxic mechanisms of carcinogenicity have also been proposed. Results of studies using the comet and Fpg modified (with foramidopyrimidine-DNA-glycosylase, an enzyme that recognizes and repairs oxidative damage in DNA) comet assays suggest that ochratoxin A causes DNA damage indirectly via oxidative stress while other studies suggest that ochratoxin A interferes with Nrf2-dependent processes in kidney that have antioxidant activity. However, evidence of oxidative DNA damage or lipid peroxidation has not been found in other studies, including experiments in which ochratoxin A was given at nephrotoxic doses (up to 2 mg/kg body weight) to male F344 rats. As is the case for the roles of genotoxicity and adduction, these hypotheses of carcinogenicity continue to be the subject of debate.

3.3. Manifestations of Toxicity in Animals Swine Swine are particularly sensitive to ochratoxin, with chronic toxicity occurring after ingestion of diets containing 0.2–4.0 ppm ochratoxin A. The kidney is the main target organ, although toxic effects following experimental exposure have been reported in the liver, lymphoid organs, and digestive tract. The disease in swine, known as porcine nephropathy, was first described in Denmark. It occurs mostly in northern Europe, but may

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be found elsewhere, including the United States. Clinically, reduced growth rates and signs of renal tubular dysfunction, including polyuria, glycosuria, and proteinuria, are observed. An early indicator of nephropathy is increased urinary leucine amino-peptidase, an enzyme present in the brush border of the proximal tubule. This increase is followed by decreased glomerular filtration rate (GFR), increased blood urea nitrogen (BUN), and decreased osmolality. Renal involvement is typically bilateral, and widespread subcutaneous, mesenteric, and perirenal edema may be present. Grossly, the kidneys may be slightly to markedly enlarged and pale, mottled or gray-white. The texture of the capsule is variable, ranging from smooth to rough, or irregular due to cyst formation. The microscopic pathology is non-specific, but is consistent with the pathology found in other species and humans with Balkan Endemic Nephropathy (BEN). There is proximal tubular degeneration and atrophy with interstitial fibrosis and mononuclear infiltration. As the disease progresses, cystic dilation of degenerated tubules occurs and, like in BEN, glomerular hyalinization can be present in severe cases. Morphologic findings alone are insufficient to diagnose ochratoxin-induced porcine nephropathy, as other agents, such as the mycotoxin citrinin, induce similar histologic changes and clinical signs. Poultry Poultry are more sensitive to ochratoxin A than to aflatoxin B1 or T-2 toxin, as growth impairment occurred in young broiler chickens fed 2 ppm ochratoxin A, as compared to 2.5 ppm aflatoxin B1, and 4 ppm T-2 toxin. The principal effect of exposure to ochratoxin A is nephropathy, but neurological and intestinal effects and visceral gout may be observed. There is a decrease in serum carotenoids and carotenoid pigmentation (leading to the loss of yellow color from the fat), which decreases marketability of the meat. Multifactorial coagulopathy leading to hemorrhage in the proventriculus and elsewhere, as well as decreased bone strength, have been reported. Also, reduced food consumption and production of eggs with substandard, stained, or rubbery shells may be observed.

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Laboratory Animals Ochratoxin A is toxic and a potent renal carcinogen in rats and mice. The kidney is the major target organ in both species. Male Dark Agouti (DA) rats appear to be more sensitive to the renal carcinogenic effect than male Lewis rats, the latter are more sensitive than female Lewis rats to ochratoxin A, while female DA rats are resistant. Among DA and Lewis rats, sensitivity to tumorigenicity by strain and sex has been correlated with both the formation of uncharacterized renal DNA adducts and the animals’ ability to metabolize debrisoquine to 4-hydroxydebrisoquine (a cytochrome P450-dependent reaction) as discussed above. Microscopic lesions were found in Fischer 344 (F344) rats orally exposed (5 days/week) to 70 or 210 mg/kg ochratoxin A, but not 21 mg/kg bw by gavage. Lesions were similar to those found in DA and Lewis rats treated by gavage; however, males were more sensitive than females. Nephropathy is characterized by the simultaneous presence of degenerative and regenerative changes in the S3 segment of the proximal tubules of the outer stripe of the outer medulla. Marked karyomegaly and cytomegaly of tubular epithelial cells which distorts the parenchymal cytoarchitecture is a characteristic feature (Figure 39.5A). Other features include degeneration and necrosis of single epithelial cells, sloughing of apoptotic cells into the tubular lumena, tubular dilation and formation of cysts, dilated tubules lined by a hyperplastic, multilayered epithelium, and tubular atrophy. If the dose is sufficiently high, nephropathy may develop in several days to weeks. Cell proliferation in the proximal tubules and medullary rays, including the karyomegalic cells, is readily demonstrated by 5-bromo-20 -deoxyuridine (BrdU) immunohistochemical staining. Ochratoxin A is nephrocarcinogenic when given to F344 rats by gavage or via the diet. Neoplastic lesions, including renal tubular adenomas and carcinomas, have been found after prolonged exposure, and multiple primary tumors were found in individual animals. Adenomas were generally well differentiated and circumscribed, resembling hyperplastic tubules. However, the proliferating neoplastic cells often obliterated the lumen and other tubular features. Carcinomas tended to be larger lesions and, in

FIGURE 39.5 Renal changes induced in F344 rats by chronic oral exposure to ochratoxin A at 210 mg/kg. H&E stain. (A) Renal epithelial cell hypertrophy and hyperplasia in the pars recta. Note the presence of enlarged epithelial cells, karyomegaly (arrow) and dilation of the tubular lumen (80). (B) Renal tubular carcinoma. Carcinomas occurred more frequently in males than females. Carcinomas were large, poorly circumscribed, showed cellular atypia, and metastasized (50). Photomicrographs provided courtesy of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC.

contrast to the adenomas, were not well circumscribed (Figure 39.5B). They had an aggressive appearance and displayed a considerable degree of cellular atypia and numerous mitotic figures. Areas of necrosis suggested that these masses expanded rapidly, outgrowing their blood supply. Metastases were readily demonstrable. A low incidence of transitional cell carcinomas or benign transitional cell papillomas of the urinary bladder also may be found in rats treated with ochratoxin A.

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In B6C3F1 mice, diets containing 40 ppm ochratoxins (about 90% ochratoxin A and 10% ochratoxin B) caused nephropathy characterized by cystic dilation and hyperplasia of tubules in both sexes. When fed for up to 2 years, renal tubular adenomas and carcinomas were found only in males. Both carcinomas and adenomas contained solid and tubular forms. No metastases were reported. Neoplastic lesions of tissues other than the urinary tract also have been described in rodents exposed to ochratoxin A for up to 2 years: ochratoxin A increased the incidence of mammary gland fibroadenomas in female F344 rats and hepatic carcinomas in female B6C3F1 mice.

3.4. Human Risk and Disease Ochratoxin A and the structurally related mycotoxin, citrinin, are the suspected etiological agents of BEN. Ochratoxin A has been placed in Group B (possibly carcinogenic to humans) by the International Agency for Research on Cancer (IARC) based upon there being sufficient evidence of carcinogenicity in animals but inadequate evidence for it in humans. The provisional tolerated weekly intake set by the Joint Expert Committee on Food Additives and Contaminants (JECFA) is 100 ng/kg body weight. As its name implies, BEN is found in a geographical area that includes parts of Serbia, Croatia, Bosnia and Herzegovina, Rumania, and Bulgaria. BEN occurs in distinct foci that are characteristically found in rural areas and are comprised of affected households irregularly interspersed among unaffected households. Although unequivocally involving specific households, heredity does not appear to play a major role. Rather, most evidence suggests an environmental etiology, including the observation that the disease occurs in outsiders settling into the endemic area. Ochratoxin A has a worldwide distribution; however, both ochratoxin A and ochratoxin Aproducing fungi are particularly common in the endemic area, and ochratoxin A concentrations of grain and foodstuffs repeatedly have been shown to be high in this region. Furthermore, both the frequency and concentration of ochratoxin A in blood or urine from BEN patients are significantly higher than those found outside

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the endemic area. For example, exposure in Canada was found to be frequent but occurring at a generally low (< 4 ng/kg body weight, all age–sex groups evaluated) level. Exposures were higher in small children. Clinically, BEN is difficult to diagnose because of its insidious onset and protracted course. Although primarily a kidney disease, liver involvement has been reported in some cases. In the early stages, there may be fatigue, pallor, headache, weight loss, pain in the loins, and proteinuria. The clinical course is one of slowly progressing chronic renal failure with disturbances in urine volume regulation, acid–base and electrolyte balance, and waste product excretion. As the disease progresses, there is azotemia, uremia, and, in some patients, hematuria or hypertension. The nephrotic syndrome of hypoalbuminemia, proteinuria, edema, and hyperlipidemia is not a feature of BEN. The characteristic finding of BEN at autopsy is bilateral atrophy of the kidneys. The microscopic lesions of BEN and porcine nephropathy, caused by ochratoxin A-contaminated feeds, are similar. Histopathology of BEN is variable, depending on the stage of the disease. Lesions are found mainly in the cortex, are usually multifocal, and involve the interstitium, vasculature, and renal tubules. More generalized involvement may be found in the end stages of the disease. Specific histological features include interstitial fibrosis, interstitial mononuclear cell infiltrates, multifocal atrophy of the proximal tubules, and vascular hyalinosis and sclerosis. Though considered a tubular disease, segmental or global thickening of the glomerular vasculature may be present and, during the end stage of the disease, may progress to overt glomerular sclerosis or hyalinization in some cases. A high incidence of urinary tract cancers, especially urotheliomas and renal cell carcinomas, has been associated with BEN. Tumors occur in patients independent of clinical signs of renal failure. The role of ochratoxin A as an etiological agent for BEN remains controversial. It is noteworthy that the morphology and anatomic location of tumors associated with BEN differ significantly in some respects from those found in rodent carcinogenicity studies. Furthermore, there is a rapidly growing body of evidence implicating aristolochic acids, which are constituents of

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39. MYCOTOXINS

Aristolochia clementis seeds that can contaminate wheat, as the etiological cause of BEN. In this regard, there are similarities in the clinical and morphological presentations of BEN and the nephropathy, including the development of tumors of urothelial origin, known to be associated with aristolocholic acid exposure. The identification of aristolocholic acid derived–DNA adducts and A to T point mutations in the TP53 tumor suppressor gene in kidneys of BEN patients has been reported.

3.5. Diagnosis, Treatment, and Prevention In animals, ochratoxicosis may be tentatively diagnosed based on clinical signs of polydipsia and polyuria as well as renal and other lesions. A definitive diagnosis is confirmed by detection of ochratoxin in toxic concentrations in foodstuffs, and enzyme linked immunosorbent assay (ELISA) kits for this purpose are now commercially available. Experimentally, ochratoxina can be detected in kidney, liver, and skeletal muscle, and ochratoxin in urine and feces. There is no specific treatment for ochratoxicosis. Supportive care should be similar to that for other causes of renal failure. Ammoniation of ochratoxin-containing grain is very effective in reducing its toxicity. Prevention of ochratoxicosis is aimed at reducing exposure to contaminated feed to the extent possible by proper agronomic, harvesting, and storage practices. Chemical and physical methods to reduce exposures through detoxification or sequestration (reduction of bioavailability) have not been rigorously explored. A provisional tolerated weekly intake of < 100 ng/kg body weight has been recommended by the World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA). The European Union (EU) has issued maximum allowable levels for ochratoxin A in foodstuffs. Values differ by commodity or product, but are (with a few exceptions for spices) 10 mg/kg or less.

4. TRICHOTHECENE MYCOTOXINS 4.1. Sources/Occurrence Trichothecene mycotoxins are a family of tetracyclic sesquiterpenoid substances (12,13

epoxytrichothecenes) comprising over 200 compounds of widely varying toxicity. They are broadly divided into two groups, the macrocyclic and non-macrocyclic trichothecenes, based on the presence or absence of a macrocyclic ring linking C-4 and C-15 with diesters (roridin series) or triesters (verrucarin series). Field fungi of the genus Fusarium are the primary producers of non-macrocyclic trichothecenes, such as T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS, anguidine), deoxynivalenol (DON), and nivalenol. The macrocyclic trichothecenes, such as roridins, verrucarins, and satratoxins, are produced primarily by Stachybotrys and Myrothecium spp. Other genera that have been reported to produce trichothecenes include Trichoderma, Trichothecium, Cephalosporium, Cylindrocarpon, Verticimonosporium, and Phomopsis. Zearalenone, while not a trichothecene, is frequently found in grains contaminated by trichothecenes. The non-macrocyclic trichothecenes are of major economic importance in agriculture due to loss of grain exports and swine production. DON is the most important trichothecene in this group, followed by T-2 and HT-2 toxins. These non-macrocyclic trichothecenes occur worldwide in grains, such as corn, wheat, barley and oats, and other commodities grown in cooler climates, including soybeans. No-till farming practice and inappropriate crop rotations, as well as the results of climate change, increase the occurrence of Fusarium. Colonization and toxin production by Fusarium spp. occur in the field, but fungal growth and toxin production can continue in storage. The disease caused by Fusarium spp. in grains is called “head blight.” Temperature and water activity are the most important factors in fungal growth, while oxygen, environmental pH, osmotic tension, and to some extent temperature, are important in toxin production. Mild temperatures tend to encourage fungal growth and cool temperatures increase toxin production (0–15
C). Trichothecenes can be found in toxic concentrations in years when cool weather conditions are followed by heavy rainfall and harvests are delayed or prolonged, such as in western Canada. DON is the most commonly detected trichothecene in cereal grains worldwide. It is produced primarily by Fusarium graminearum (Gibberella zeae, Fusarium roseum) and Fusarium

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4. TRICHOTHECENE MYCOTOXINS

culmorum, with corn, oats, barley, and wheat being the most important sources of exposure. 3-Acetydeoxynivalenol and 15-acetydeoxynivalenol, acylated forms of DON, are also frequently present in contaminated grains. T-2 and HT-2 toxins are produced primarily by Fusarium sporotrichioides, with highest concentrations generally occurring in small grains such as barley and wheat. Corn and forages can be contaminated. T-2 and HT-2 toxins are generally present together in contaminated grain, with HT-2 toxin concentration representing two-thirds the sum of T-2 and HT-2 toxin concentration. Trichothecenes can cause adverse effects in humans consuming grain-based foods and in animals ingesting contaminated grain or hay. Inhalation or dermal exposure can also induce toxicity in some cases. Trichothecene toxicosis is manifested by a broad spectrum of clinical disorders that vary according to the specific toxin or mixture of toxins present in contaminated feed or the environment. Species differences in response are generally related to severity of the response, and young animals are more susceptible than adults. In the USA and Canada, DON appears to be the most significant member of the group despite its comparatively low toxicity. Losses in swine production are the most common manifestation of toxicity in North America and Europe, while cases of human poisoning with gastrointestinal manifestations have been reported from China and India. T-2 toxin has been implicated in widespread epidemics of alimentary toxic aleukia (ATA) in humans in the Soviet Union that occurred in the 1930s and 1940s. This was based on the retrospective isolation of Fusarium sporothrichioides and F. poae from grain associated with the disease outbreaks. Both T-2 toxin and DAS have occasionally caused outbreaks of toxicoses in animals in North America and Japan. T-2 toxin and DAS, along with DON and zearalenone, were detected in specimens of the alleged chemical warfare agent “Yellow Rain” in Southeast Asia. Macrocyclic trichothecene toxins are not produced by Fusarium spp., but by fungi such as Stachybotrys chartarum (Stachybotrys atra), a black fungus growing in wet forages and/or straw as well as in water-damaged building materials and water-soaked air ducts. These macrocyclic trichothecenes, whether ingested

1209

or inhaled, can cause adverse health effects in animals and humans. Inhalation of these mycotoxins has been proposed as one cause of the “sick building” syndrome, and recent research indicates that they have the potential for both respiratory and neurotoxicity. An edible fungus, Podostroma cornu-damae, found in Japan and China, also produces these toxins and has been responsible for several poisonings.

4.2. Toxicology Toxin All trichothecene mycotoxins have a basic tetracyclic sesquiterpene structure with a sixmembered oxygen-containing ring, an epoxide group in the 12,13 position, and an olefinic bond in the 9,10 position (Figure 39.6). They have been classified into four groups based on substitutions at five positions of the trichothecene skeleton (Figure 39.6). Group A trichothecenes possess hydroxyl or esterified hydroxyls at the C-3, 4, 7, 8 or 15 positions, and include T-2 toxin, HT-2 toxin, DAS, and monoacetoxyscirpenol. Group B trichothecenes contain a keto group at C-8 and a hydroxyl group at C-7, in addition to other functional groups, and include DON and nivalenol. Group C trichothecenes typically have a second epoxide ring at C-7, 8, and include crotocin. Group D trichothecenes have a macrocyclic ring linking C-4 and C-15 and include macrocyclic trichothecenes, such as verrucarins and satratoxins produced by Myrothecium and Stachybotrys spp., respectively. While fungi frequently produce several toxins, most species tend to produce toxins from one of the groups A, B, or D. An example of a fungus that produces several different types of toxins is Fusarium graminearum. It is a primary producer of zearalenone, but can also synthesize both group A and B trichothecenes. Biodistribution, Metabolism, and Excretion Trichothecenes can be absorbed through the gastrointestinal and respiratory tracts, as well as skin. Most of the information on toxicokinetics of trichothecenes is based on iv administration because of feed refusal evidenced in all species and the powerful emetic effects in larger laboratory species and domestic animals. Thus limited information is available on oral and inhalation

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FIGURE 39.6 General chemical structure of trichothecenes and mycotoxins from groups A (T-2 toxin) and B (deoxynivalenol).

routes of exposure. Radiolabeled studies with the trichothecene skeleton (Figure 39.6) as well as individual toxins indicate rapid absorption, distribution, and excretion following oral or parenteral administration. Trichothecenes do not accumulate in the body to any great extent, and residues are readily excreted within several days following exposure. One exception is the slow absorption of T-2 toxin when applied dermally, with skin and subcutaneous fat acting as reservoirs for the toxin, resulting in delayed absorption and sustained metabolism and excretion. Comparison of oral and iv routes of administration of the trichothecene skeleton indicates a first-pass effect of the liver. The trichothecenes can undergo phase 1 (hydrolysis, oxidation, reduction) and 2 (glucuronide conjugation) biotransformation. Specific metabolic pathways of these mycotoxins differ, and the metabolites produced often differ among species. The majority of these reactions occur in tissues and result in reduced toxicity; however, some metabolites may be more toxic than the parent. Significant de-epoxidation by rumen microorganisms occurs. Excretion is via the biliary system and urine. Enterohepatic recirculation may occur, especially in swine, resulting in delayed excretion and, ultimately, increased

toxicity. It should be noted that HT-2 toxin is a major metabolite of T-2 toxin. Because of the importance of DON to the swine industry, the toxicokinetics of DON have been extensively studied in this species. Intravenously administered DON (1 mg/kg body weight) was distributed rapidly to all tissues and body fluids, and declined to negligible levels within 24 hours except for urine and bile. The half-life of DON (0.5 mg DON/kg bw iv) in swine is between 2.08 and 3.65 h, suggesting that 97% would be eliminated in 10.1–18.3 h. DON concentrations decreased biphasically with terminal elimination half lives (t1/2 b) of between 4.2 and 33.6 h. Orally administered DON is rapidly and nearly completely absorbed from the stomach and proximal small intestine. Pigs given DON in one oral dose or via the diet (5.7 mg/kg diet) for 4 weeks rapidly absorbed  50% of administered DON. The toxin rapidly left the blood (apparent volume of distribution exceeded the total body water), with serum elimination halflives of 5.3 and 6.3 h respectively and total elimination times (97%, five elimination half-lives) of 26.5 and 31.5 h, respectively. The majority of the DON was eliminated via the urine and feces, with urinary excretion of unmetabolized DON

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4. TRICHOTHECENE MYCOTOXINS

accounting for most of the compound. DON was not significantly transferred into milk, meat, or eggs. Mechanism of Action Trichothecenes inhibit synthesis of protein, RNA, and DNA as well as mitochondrial and electron chain function, stimulate lipid peroxidation, alter cell membrane function, induce apoptosis, modulate immune responses, activate mitogen-activated protein kinases (MAPKs), induce gene expression of numerous chemokines and cytokines, and alter neurotransmitter levels. All trichothecenes target the 60S ribosomal subunit, suggesting that the major mechanism of toxicity is translational inhibition. Inhibition of protein synthesis occurs through interference with peptidyl transferase activity, with an intact C-9,10 double bond and the C-12,13 epoxide required for this inhibition. Inhibition takes place in the translational stage that occurs in the polysomes of the endoplasmic reticulum. All three translational processes – initiation, elongation, and termination – can be affected; however, the inhibitory potency and major site of action during translation vary among trichothecenes. Trichothecenes with hydroxyl and acetyl substitutions at both C-3 and 4, such as T-2 toxin, DAS, and verrucarin A, preferentially inhibit initiation while DON, trichodermin, crotocin, and verucarol inhibit elongation and/or termination. Trichothecenes and other translational inhibitors that bind to ribosomes also rapidly activate mitogen activated protein kinases (MAPKs) and induce apoptosis in a process known as the “ribotoxic stress response.” MAPKs modulate processes such as cell growth, differentiation, and apoptosis, and are critical for signal transduction in the immune response. Trichothecene-induced MAPK activation mediates pro-inflammatory cytokine upregulation in addition to apoptosis. It is now recognized that apoptosis is a major mechanism for toxicity induced by DON, T-2 toxin, and other trichothecenes. Apoptosis can be driven by both extrinsic (death receptormediated) and intrinsic (mitochondrial mediated) pathways. Some biological effects of trichothecenes are mediated by reaction of the epoxy groups of trichothecenes with sulfhydryl groups on enzymes and binding of certain trichothecenes to

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membrane components. Other effects of trichothecenes may be mediated via lipid peroxidation. Recent studies indicate that some trichothecenes can bind covalently to cell macromolecules – for example, satratoxin G, a macrocyclic trichothecene that forms covalent adducts with proteins and potentially other macromolecular targets. The ability of satratoxin G to bind to albumin provides a potential biomarker of exposure to the toxin as well as to Stachybotrys chartarum. Trichothecenes can be immunostimulatory or immunosuppressive depending on dose, exposure frequency and timing relative to sampling for immune assays. Low-level exposure promotes, in hormetic fashion, the expression of numerous pro-inflammatory cytokines and chemokines. Both transcriptional and posttranscriptional mechanisms are involved in trichothecene-induced cytokine mRNA expression. One example of cytokine upregulation is DON-induced IgA dysregulation in mice, which results in glomerulonephritis that closely resembles human IgA nephropathy. Induction of a “cytokine storm” could contribute to shocklike effects exhibited at near lethal doses. At high doses, trichothecenes are cytotoxic to B and T cells in lymphoid tissues including the thymus and Peyer’s patches, leukocytes of bone marrow, and macrophages, which appear to be especially sensitive. Collectively, these effects result in immune suppression, which increase susceptibility to infectious agents. Some trichothecene-induced effects may be a result of altered neurotransmitters in the central (CNS) or peripheral nervous system (PNS). Emesis (vomiting), which occurs acutely with many of the trichothecenes at high doses, had been attributed to stimulation of the chemoreceptor trigger zone in the area postrema of the medulla oblongata. However, studies with T-2 toxin in cats indicated that other mechanisms, such as the neural afferent pathways from the abdomen, implicated in radiation-induced emesis, may also be involved. The mechanism for reduced feed intake that follows chronic low-dose DON exposure has received a lot of attention since growth suppression is a public health concern, especially for infants. Changes in neurotransmitters, such as dopamine, tryptophan, and serotonin in the central nervous system, are not consistent with appetite suppression. Other hypotheses examined

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39. MYCOTOXINS

include a serotonergic effect mediated through enteric release of serotonin by enterochromaffin cells (see Digestive Tract, Chapter 56), the action of pro-inflammatory cytokines on the central nervous system, and interference with signaling pathways affecting the growth hormone axis. Recent research on DON-mediated appetite suppression in the mouse, which cannot vomit but develops DON-induced anorexia, suggests that this effect may be mediated by the release of the gut satiety hormone peptide YY (PYY) and cholecystokinin (CCK) in the intestinal tract. These hormones, released from enteroendocrine cells, can promote signaling changes within the hypothalamus by affecting anorexigenic and orexigenic peptides. Insufficient data are available to draw a firm conclusion regarding the mutagenicity or carcinogenicity of the trichothecenes. Trichothecenes are not mutagenic in bacterial assays, although DON induced chromatid breaks in Chinese hamster cells (CHO) and T-2 toxin was genotoxic in several tests at cytotoxic dose levels. Carcinogenesis bioassays have been largely negative, although T-2 toxin induced hepatocellular and pulmonary adenomas in male CD1 mice. Evaluation of available data by IARC has concluded that the trichothecenes are not classifiable as to their carcinogenicity to humans (Group 3). Trichothecenes have not been shown to be teratogenic in mammals except at maternally toxic doses at which embryo lethality and anomalies in the nervous and skeletal systems have been observed. Toxicity and Species Susceptibility Median lethal dose (LD50) values, which may differ based on route of exposure and species exposed, can be used to compare the toxicity of trichothecenes (Table 39.5). For example, the LD50 values for ip exposure in the mouse are 5.2 mg/kg for T-2 toxin and 23 mg/kg for DAS, while for iv exposure in the pig they are 1.21 mg/kg for T-2 toxin and 0.37 mg/kg for DAS. For T-2 toxin, oral LD50 values are not markedly different across species; however, T-2 toxin was 10- to 50-fold more toxic when inhaled than when administered orally. HT-2 toxin is less toxic than T-2 toxin, while DON is one of the least lethal trichothecenes. The pig is one of the most sensitive species to all trichothecenes. While human data are limited, the anatomical

and physiological similarity of pigs and humans should be considered in extrapolation of data across species. Recent research has focused on DON due to its common occurrence in cereal-based food. All monogastric species, such as pigs, dogs, cats, mice, and rats, are sensitive to DON-induced reduction in weight gain following chronic lowlevel exposure, with pigs being the most sensitive. In pigs, the NOEL is < 1 ppm, while in dogs the NOEL is 3–6 ppm. Ruminants, horses, rabbits, and poultry are relatively resistant species. Cats are very extremely sensitive to T-2 toxin, with mortality observed at 60–100 mg/kg bw per day orally. This is presumably due to their inability to excrete T-2 toxin and its metabolites via glucuronide conjugation. Using immunological or hematological effects as endpoints, the lowest adverse effect level (LOAEL) in the pig is 29 mg/kg bw per day. Some additional LOAELs are 40 to 48 mg/kg bw per day in poultry, 100 mg/kg bw per day in humans, and 300 mg/kg bw per day in young ruminants. The no observed adverse effect level (NOAEL) for catfish is 13 mg/kg bw per day and for rabbits is 100 mg/kg bw per day. No toxicity data are available for dogs, and neither NOAEL nor LOAEL is available for horses.

4.3. Manifestations of Toxicity in Animals General Reduced feed intake, feed refusal, and emesis are associated with most trichothecenes, with swine being the most sensitive species. Decreased feed consumption and feed refusal leads to reduced weight gain and production loss. Emesis is an acute reaction that occurs rapidly after either oral or parenteral administration. The more potent trichothecene mycotoxins, T-2 toxin, DAS, and the macrocyclic toxins induce cytotoxic effects due to direct local contact with skin and oral mucosa; DON, in contrast, does not. T-2 toxin, DAS, and the macrocyclic toxins, also cause hematotoxicity and immune suppression, considered to be the most sensitive adverse effects, and, in some cases, neurotoxicity. Hematologic and immunologic alterations, including leukopenia, are potentially devastating consequences of exposure.

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Acute LD50 values (mg/kg)1

Lethal dose (mg/kg)

Group

Main Producer

Trichothecene

Mouse iv or ip

Mouse oral/po

Mouse inhalation2

Pig iv

Pig inhalation3

A

Fusarium sp.

T-2 toxin

3.0e5.3

3.8e10.5

0.16

1.21

1.5e3.0

HT-2 toxin

6.5e9.0

Diacetoxyscirpenol (DAS)

9.6e23.0

15.5e46.0

70.0e76.7

46.0

0.37

Monoacetoxyscirpenol B

Fusarium sp.

Deoxynivalenol (DON, vomitoxin) Nivalenol Fusarenon X

C

Cephalosporium sp.

Crotocin

D

Myrothecium sp.

Verrucarins Verrucarin A and B Roridins Roridin A

Stachybotrys sp.

4.0e6.3 3.4

4.5

700e810

1000

0.5 (A) e7.0 (B)

4. TRICHOTHECENE MYCOTOXINS

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TABLE 39.5 Partial Listing of Trichothecene Toxins and their Comparative Toxicity

1.0 (A)

Satratoxins

1

Information largely from Trenholm et al. (1989), Ueno, 1985 24-Hour LD50, in saline solution; Cresia and Lambert (1989) 3 Estimated retained dose (20–30%) using dry aerosol of T-2 toxin, all animals died within 18 hours; Cresia and Lambert (1989). Table reproduced from Haschek and Beasley (2009). Trichothecene mycotoxins. In: Handbook of Toxicology of Chemical Warfare Agents (R. C. Gupta, ed.), Elsevier Inc., Table 26-1, p. 354, with permission. 2

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39. MYCOTOXINS

Immune suppression may be the most significant effect of low-level trichothecene exposure leading to increased susceptibility of exposed animals and humans to infectious and other diseases. Immunotoxicity can be amplified by low levels of Gram-negative bacterial lipopolysaccharide (LPS), a prototypic inflammagen; this may explain some cases of increased individual susceptibility to trichothecenes and other mycotoxins. Deoxynivalenol (DON) Though DON is not one of the more acutely toxic trichothecenes, DON-contaminated feed has caused great economic loss to the livestock, especially swine, industry due to the well-documented reduction in feed consumption and weight gain. High-dose acute DON exposure results in emesis (in sensitive animals that can vomit), diarrhea, abdominal distress, increased salivation, and listlessness, again similarly to other trichothecenes. In pigs, the main clinical effect of ingested high-dose DON is rapid-onset emesis; this depends not only on concentration of DON in the feed but also on the time period over which a given amount of feed is ingested. Chronic low-dose DON exposure causes a reduction in food intake or feed refusal (anorexia) and reduces weight gain; decreased efficiency of feed utilization has been reported. Such responses apparently limit exposure and thus toxic manifestations. With purified DON, transient effects on feed intake can be observed in swine at concentrations as low as 2–4 ppm, with more permanent effects at > 5 ppm and severe food refusal at > 20 ppm (20 mg/kg). DON in feed at 4 ppm caused a 2% reduction in feed intake, while at 40 ppm a 90% reduction was observed in one study. In the field, however, concentrations of DON associated with feed refusal may be as low as 1 ppm (considered the lowest adverse effect level), although at this low a level feed refusal may be due to the presence of other mycotoxins, known and unknown, in the ration. Other signs may include soft stools, diarrhea, failure to thrive, and a predisposition to other disease entities and poor nutrition. Emesis, which occurs at higher levels of exposure, is infrequently seen in the field; therefore, the name “vomitoxin,” encountered in the literature, is inappropriate. With repeated exposure, pigs may develop a resistance to DON and make

compensatory gains. Although mild thickening of the squamous mucosa of the stomach has been noted experimentally in swine, specific lesions are not observed under field conditions. An acute lethal dose of DON to mice induces intestinal hemorrhage and necrosis of bone marrow and lymphoid tissue similarly to other trichothecenes. Mice are a good model for studying DON-induced reduction in weight gain and anorexia, since these occur at DON levels as low as 2 ppm in the diet. As with other trichothecenes, immune dysregulation occurs, with mononuclear phagocytes likely playing a central role. Low concentrations of DON induce expression of early response and pro-inflammatory genes at the mRNA and protein levels, while high concentrations promote rapid onset of leukocyte apoptosis. Chronic dietary studies in mice have shown that DON impairs humoral immunity, cell-mediated immunity, and host resistance. In addition, serum immunoglobulin A (IgA) becomes elevated due to dysregulation of IgA production and IgA accumulates in the mesangium of the kidney. Similar results have been obtained with nivalenol in mice and pigs. This dysregulation is similar to human IgA nephropathy, globally the most common form of glomerulonephritis. The disease is progressive in up to 50% of affected individuals, resulting in renal failure. The cause of IgA nephropathy is unknown, and the possibility of mycotoxin involvement in the disease process needs to be considered. Reproductive effects in male rats ( 2.5 mg DON/kg bw by gavage) consisted of decreased body weight, sperm counts, and serum testosterone levels, and increased serum FSH and LH concentrations. Testicular germ cell degeneration and abnormal development as well as failure of sperm release were observed. T-2 Toxin and Diacetoxyscirpenol (DAS) OVERVIEW

The toxic effects of T-2 toxin and DAS are similar irrespective of route of exposure and species, with ingestion, parenteral administration, or inhalation resulting in lymphocyte necrosis, hematotoxicity, and gastrointestinal toxicity (Table 39.6, Figure 39.7). The main effects are due to necrosis of rapidly dividing cells, the so-called radiomimetic effect. Shock secondary

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TABLE 39.6

Target Organ Toxicity of T-2 Toxin and DAS

Tissue Lymphoid

Exposure route All

a

Bone marrow

Mostb

Gastrointestinal

Mostb Most

b

Clinical Signs, Hematology

Pathology

Thymus

Immune suppression, lymphopenia

Apoptosis (T cell), lymphoid depletion

Spleen, lymph nodes

Immune suppression, lymphopenia

Apoptosis, lymphoid depletion, B cell primarily

Leukopenia, anemia, thrombocytopenia

Apoptosis, necrosis e all cell types

Stomach

Vomiting, dehydration

Ulceration, parietal cell injury

Small intestine

Diarrhea, dehydration

Apoptosis, epithelial cell, both surface and crypt

Gall bladder

Mosta

Skin

On contact, dermal

Mucous membrane

On contact, oral

Oral and buccal mucosa

Pancreas (pig only)

Intravenous, inhalation, dermal

Exocrine

Gall bladder

Edema, hemorrhage Irritation, vesicles, desquamation

Necrotizing dermatitis

Oral/buccal vesicles, ulcers

Stomatitis Acinar cell necrosis

Endocrine

Hyper-or hypoglycemia

Islet cell necrosis (DAS, iv)

Heart

Decreased cardiac output and aortic blood pressure (T-2 toxin, pig, rat) Hypotension (DAS, human)

Myocardial hemorrhage and necrosis (T-2 toxin, pig iv, inhalation, and rat ip)

Cardiovascular system

Parenteral, inhalation

Central nervous system

Parenteral, inhalation

?Vomiting

None

Parenteral, inhalation

Somnolence, confusion, ataxia (DAS, humans) Dizziness, vertigo (“Yellow Rain,” humans)

Not examined

Parenteral, dermal

Ataxia, posterior paresis (pig)

Capillary endothelial cell necrosis and hemorrhage (DAS, iv)

Infertility

Necrosis of germinal epithelium

Reproductive system

Testis

Parenteral, inhalation, ingestion, dermal. Parenteral, inhalation, ingestion. Table modified from Haschek, W. M. and V. R. Beasley (2009). Trichothecene mycotoxins. In: Handbook of Toxicology of Chemical Warfare Agents, R. C. Gupta, ed. Elsevier Inc. Table 26-2, p. 359, with permission.

b

1215

a

Parenteral

4. TRICHOTHECENE MYCOTOXINS

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Alla

Organ

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39. MYCOTOXINS

FIGURE 39.7 Intestine from Sprague-Dawley rats given T-2 toxin at 25 mg/kg as a single oral dose 12 hours previously. H&E stain. (A) Duodenum from a control rat. The mucosa consists of normal elongated villi (V), crypts (C), and Brunner’s glands (B) located at the base of the mucosa (100). (B) Duodenum from a treated rat. The intestinal villi are short and blunted. The villous epithelial lining is segmentally ulcerated. Brunner’s glands and crypts are largely destroyed (100). (C) Higher magnification of B. Extensive apoptotic necrosis of crypt cells is present. Cellular debris and mononuclear inflammatory cells are present in the lamina propria (400). Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 7, p. 667, with permission.

4. TRICHOTHECENE MYCOTOXINS

to cardiovascular collapse occurs at high doses. T-2 toxin and DAS are highly irritating to the skin and mucous membranes. Toxicosis due to ingestion of feed naturally contaminated with T-2 toxin and DAS has been reported mainly in swine, poultry, and cattle, but occasionally also in horses, dogs, cats, and humans. Clinical signs most often consist of reduced feed intake or feed refusal, emesis, diarrhea, necrosis of skin and oral mucosa, and increased incidence of infection. Because T-2 toxin causes feed refusal and vomiting, severe toxicosis is rarely reported in field situations. In experimental toxicosis, the lesions of T-2 toxin and DAS include hemorrhage and necrosis of gastrointestinal mucosa (high doses), destruction of hemopoietic tissue and lymphoid necrosis (high doses), and meningeal hemorrhage (massive doses). Shock and death can follow massive doses. Less well-known effects of T-2 toxin include necrosis of the adrenal cortex, kidney, and liver, and, in pigs, pancreas and myocardium (presumably due to release of myocardial depressant factor from the injured pancreas). Clotting disorders and reproductive problems also have been reported with T-2 toxin. At doses toxic to the dam, embryotoxicity and fetotoxicity, as well as abortion, may occur. Testicular damage may occur at high doses. Dermal application results in severe skin irritation with cytotoxicity, and has been used as a bioassay for trichothecenes. T-2 toxin induced immunotoxicity occurs at both the humoral and cellular immune levels (see Immune System, Chapter 49). Evidence of cytotoxicity is observed as apoptosis of lymphocytes and hematopoietic cells. CD34þ hematopoietic progenitor stem cells and CD4þ and CD8þ lymphocyte subsets are particularly affected. Dendritic cells are sensitive to trichothecenes, and topical application of T-2 toxin inhibits activity of Langerhan’s cells. Antibody production is suppressed. Inhibition of inflammatory cell function and decreased immune responses by T-2 increases susceptibility to bacterial and viral infection. SWINE

At the LD50 dose or above, pigs develop a shock syndrome, with death in 12–16 hours. Acute effects include vomiting, lethargy, frequent defecation and diarrhea, and posterior

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paralysis. Surviving pigs recover and appear normal within 24 hours. Hematologic effects include a transient leukocytosis due to neutrophilia, followed by leukopenia due to lymphopenia and neutropenia; cell-mediated and humeral immune responses are depressed. Systemic toxicity is manifested by injury to the gastrointestinal tract, especially stomach and small intestine, and the immune and hematopoietic systems. Gastric toxicity is characterized by hemorrhagic ulcerative gastritis, whereas ulceration of the surface mucosa and selective necrosis of crypt epithelial cells characterizes the lesions in the intestinal tract (Figure 39.7). Lymphocyte necrosis occurs in the thymus, lymph nodes, spleen, and other lymphoid tissues, with B lymphocytes more severely affected than T lymphocytes. Necrosis of hematopoietic cells in the bone marrow is also present. The selective targeting of rapidly dividing cells, such as the intestinal crypt epithelial cells, as well as those in the immune and hematopoietic systems, underlies the so-called “radiomimetic” effect ascribed to T-2 toxin and DAS (see radiation effects described in Radiation and Other Physical Agents, Chapter 44). Toxicity to the heart, pancreas, as well as adrenal cortex, liver, and kidney also has been reported. Transient pulmonary inflammation occurs following experimental inhalation exposure, as does severe dermatonecrosis (Figure 39.8) following dermal exposure, both presumably due to direct cytotoxicity. Most non-respiratory effects following inhalation exposure were similar to those following iv exposure. With dermal exposure, local effects were marked but systemic effects were much less severe and limited to the lymphoid system and pancreas. Dietary exposure leads to similar but less severe hematotoxic and immunotoxic effects (at  0.5 mg/kg), as well as decreased feed intake leading to decreased body weight gain (at  1 mg/kg). Direct cytotoxic effects to the snout and oral cavity can be seen at higher doses. RUMINANTS

Calves and lambs exposed to  0.3 mg T-2 toxin/kg bw per day (considered the LOAEL) develop gastrointestinal lesions, altered serum proteins, and altered hematological and immune functions. T-2 toxin fed to calves at 10–50 ppm caused necrosis of the oral mucosa, ruminal

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reported. Other clinical signs include abnormal feathering; reduced feed efficiency and growth suppression; decreased egg production, eggshell thickness, and shell strength; fecal urate crystals; and diarrhea.

FIGURE 39.8 Skin from a pig given T-2 toxin at 15 mg/kg as a single dermal application. (A) At 3 days after application the area was markedly reddened and edematous. (B) At 1 day after application there is multifocal ballooning degeneration of the epidermis, as well as edema and mild cellular infiltration around congested vessels in the dermis (H&E, 10). Figure 39.8(B) reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 9B, p. 669, with permission.

and abomasal ulcers, severe diarrhea, and thymic atrophy. Adult ruminants are considered less sensitive than other species because of detoxification of T-2 toxin in the rumen. POULTRY

Oral ulceration, nervous signs, hepatic hematoma, and reduced weight gain occurred in broilers fed diets containing 4 ppm T-2 toxin or DAS. At higher dietary concentrations, hematopoietic damage and coagulopathy were

Macrocyclic Trichothecenes (Stachybotryotoxicosis) Stachybotrys chartarum (Stachybotrys atra), the main fungus associated with stachybotryotoxicosis and sick building syndrome, is a black mold. There are two toxic “chemotypes” of S. chartarum, one elaborating highly toxic macrocyclic trichothecenes, such as satratoxins, roridin, and verrucarin, and the other chemotype producing the less toxic compounds, atranones and the simple but not macrocyclic trichothecenes. Myrothecium and Dendrodochium spp. can also produce macrocyclic trichothecenes. Exposure may be by direct contact, ingestion, or inhalation. Stachybotryotoxicosis occurs after ingestion of moldy straw or hay contaminated with these highly toxic macrocyclic trichothecenes. It has been reported in horses, ruminants, swine, and poultry in the former Soviet Union and adjacent countries, as well as Finland, Hungary, France, and South Africa. Macrocyclic trichothecenes are directly toxic to mucosal membranes, causing necrosis and edema of the lips, tongue, and buccal membranes, and later diarrhea due to gastrointestinal toxicity. Hematopoietic toxicity follows and is characterized by leukopenia, thrombocytopenia, and coagulopathy, which can result in systemic hemorrhage, septicemia, and death. These effects are similar to those induced with T-2 toxin and DAS. Horses consuming large amounts of contaminated feed may show nervous signs and evidence of circulatory collapse, dying in 1–3 days. This has been termed the “shocking” form of stachybotryotoxicosis. Affected horses may experience hyperesthesia, hyperirritability, blindness, stupor, a wide stance, crossed legs, difficulty swallowing, an atonic intestinal tract, diarrhea, shock, and cyanosis. At necropsy, lesions include widespread hemorrhages (evidence of hemorrhagic diathesis), ulceration of mucosa of the alimentary tract, pneumonia, renal infarcts, multifocal hepatic necrosis, bone marrow necrosis, and lymphoid depletion. Some of these lesions may be due to secondary infections.

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Humans with direct contact or following inhalation of material infected with S. chartarum may develop dermal toxicity as well as respiratory distress, epistaxis, and eye irritation. It is generally accepted that buildingrelated asthma and an increased incidence of respiratory symptoms are associated with living or working in a “moldy” environment, which is one cause of the so-called “sick building” syndrome (see Section 4.4, on human risk). Neurocognitive dysfunction, mucous membrane irritation, and immune disorders have also been reported.

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The macrocyclic trichothecenes satratoxin G, isosatratoxin F, and roridin A cause nasal and pulmonary toxicity when administered intranasally or intratracheally to mice. Intranasal exposure of satratoxin G and roridin A induced apoptosis of olfactory sensory neurons, resulting in atrophy of the olfactory epithelium and olfactory nerve layer of the olfactory bulb in the frontal brain of mice; similar changes were induced with satratoxin G in rhesus monkeys (Figure 39.9). Marked changes in surfactant synthesis and secretion were found in alveolar type II cells and alveolar macrophages following

FIGURE 39.9 Satratoxin G (SG)-induced atrophy of olfactory epithelium. (A, B, C) Photomicrographs of the olfactory epithelium (OE) lining the dorsal nasal septum of a rhesus monkey treated with saline alone in the left nasal passage (A, B), and repeated low-dose SG (5 mg per day  4 days) in the right nasal passage (A, C). Note the presence of apoptotic olfactory sensory neuron (OSN) nuclei (arrows) and epithelial atrophy in the SG-exposed nasal passage (C). NB, olfactory nerve bundles; SB, septal bone; dashed line, basal lamina. H&E stain. Figure reproduced from Carey et al. (2012) Satratoxin-G from the black mold Stachybotrys chartarum, Toxicol. Pathol. 40, Fig. 2, p. 891, with permission.

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intratracheal instillation of isosatratoxin F or Stachybotrys spores.

4.4. Human Risk and Disease Introduction Acute high-level dietary exposure to trichothecenes should be considered in foodborne disease outbreaks that present as gastroenteritis, especially in developing countries. In addition, chronic low-level trichothecene exposure should be considered as a potential cause of growth suppression and immune dysregulation, resulting in increased susceptibility to infectious and other diseases. Inhalation exposure to trichothecenes should be considered in people with “sick building” syndrome and those living in moldy environments showing respiratory and/or neurologic signs. Human diseases that may be linked to trichothecene exposure based on research data include IgA glomerulonephritis (DON) and Kashin-Beck osteoarthritis (T-2 toxin). Risk assessment of dietary exposure for humans is difficult due to limited availability of toxicokinetic and toxicodynamic data and the paucity of recent cases of human illness reported from consumption of food derived from heavily contaminated grains. Risk assessment from non-dietary routes of exposure in humans is also complicated by lack of epidemiological data (see Risk Assessment, Chapter 31). Deoxynivalenol Human exposure to DON is primarily through ingestion of cereals and grains. In the USA and Europe the main source is wheat, while in the Far East wheat and other grains contribute equally; corn also contributes to exposure. DON has been implicated in outbreaks of acute human mycotoxicoses occurring in India, Japan, Korea, and China, with thousands of people affected in some outbreaks. DON was found in samples of wheat products associated with these outbreaks, ranging from less than 10 ppm to 93 ppm as upper limits. In the 1984–1985 outbreaks in China, a large number of people who had ingested moldy corn and wheat containing DON and zearalenone developed gastrointestinal symptoms including nausea, vomiting, abdominal pain, diarrhea, dizziness, and headache within 5–30 minutes of ingestion.

Of more concern in developed countries are potential subtle effects of chronic low-dose DON exposure in the young and old, as well as vegetarians. The availability of new biomarkers to determine exposure (e.g., DON glucuronide) and effect (e.g., insulin-like growth factor 1, IGF1) will allow future epidemiological studies to determine whether there is a link between low-level DON exposure and adverse effects in sensitive human populations. The US FDA has set an advisory level for DON of 1 ppm in finished wheat products, such as flour, germ, and bran, for human consumption. The EU has somewhat more conservative regulations. The tolerable daily intake (TDI) for DON in the European Union is 1 mg/kg bw per day. For animal feed, the US FDA advisory level is 10 ppm for beef cattle (> 4 months of age) and chickens, and 5 ppm for swine (< 20% of ration) and all other animal species (< 40% of ration) T-2 Toxin and DAS Toxins produced by Fusarium sporotrichoides, with T-2 toxin believed to be the principal toxin, were implicated in the human disease, alimentary toxic aleukia (ATA). This was based on the retrospective isolation of Fusarium sporothrichioides and F. poae from grain associated with the disease outbreaks. Alimentary toxic aleukia was recognized prior to 1900 in the former Soviet Union, where thousands of people developed ATA and many died after consuming cereals that overwintered in fields during and after World War II. The symptoms of ATA include fever, vomiting, acute inflammation of the entire alimentary tract, anemia, sepsis, circulatory failure, and convulsions. Death often followed. T-2 toxin, together with DON, nivalenol, and deoxynivalenol monoacetate, was isolated from moldy flour used to make bread that was associated with an outbreak of human illness in Kashmiri, India in 1987. The major symptoms were abdominal pain, inflammation of the throat, diarrhea, vomiting, and melena (bloody stools). Human exposure to trichothecene mycotoxins was also alleged to occur in Southeast Asia and Afghanistan from exposure to a chemical warfare agent named “Yellow Rain.” T-2 toxin, DON, nivalenol, and DAS, implicated as components of “Yellow Rain,” were detected in low concentrations in blood, urine, and tissue samples

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of alleged victims. Clinical signs included vomiting, diarrhea, headache, fatigue, dermatitis with focal alopecia, and generalized malaise. T-2 toxin has been recently identified as a potential cause of Kashin-Beck disease, a form of endemic osteoarthritis, in China. Epidemiologic studies indicate a possible link to trichothecene exposure, and rats fed T-2 toxin had degenerative changes in articular cartilage similar to those seen in Kashin-Beck disease. In the 1970s, DAS (anguidine) underwent Phase 1 and 2 clinical trials as a treatment for cancer. Although minimal antitumor activity was reported, these studies allowed documentation of clinical signs and hematopoietic effects of trichothecenes in humans. Clinical signs included nausea, vomiting, diarrhea, hypotension, and CNS disturbances. Myelosuppression was also observed. Grains and grain-based products are the main source of exposure for T-2 and HT-2 toxins. These toxins are relatively stable during baking and cooking, and during manufacture of compound feedstuffs. The TDI for humans in the EU is 100 ng/kg bw for the sum of T-2 and HT-2 toxins. JECFA derived a provisional mean (PM) TDI of 0.06 mg T-2 and HT-2 toxins/kg bw per day for pigs. EFSA (2011) has estimated the exposure values at the lower and upper bound concentrations for the sum of T-2 and HT-2 toxins in diets for a number of livestock and companion animal categories based on expected feed intakes and example diets. Macrocyclic Trichothecenes Stachybotryotoxicosis is well recognized in humans in the former Soviet Union and adjacent countries following contact with macrocyclic trichothecenes produced by Stachybotrys chartarum and other non-Fusarium spp. on moldy straw or hay. Farm workers in contact with infected litter or feed developed skin rashes, respiratory distress, epistaxis, and eye irritation. A more recent concern for macrocyclic trichothecene exposure is the presence of Stachybotrys chartarum and, less often, other non-Fusarium spp. in water-damaged buildings or air ducts (one cause of the so-called “sick building syndrome,” “damp building related syndrome,” or “damp building related illness” [DBRI]). Clinical signs include vomiting, diarrhea, headache, fatigue, dermatitis with focal alopecia, and

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generalized malaise. One study from Montreal, Canada, identified toxins, including T-2 toxin, DAS, roridine A, and T-2 tetraol, in a “sick” building. Experimental studies in mice and monkeys suggest that nasal inflammation, mucus hypersecretion, and olfactory neurotoxicity could be important adverse health effects associated with short-term, repeated airborne exposures to macrocyclic trichothecenes. The possible involvement of exposure to such mycotoxins in asthma is an issue since there is an association between fungal exposure and increased bronchial responsiveness (see Respiratory System, Chapter 51). Additionally, idiopathic pulmonary hemorrhage in infants, which can be fatal, has been attributed by some to exposure to fungi and their toxins, especially to Stachybotrys chartarum that grows in water-damaged homes.

4.5. Diagnosis, Treatment, and Prevention General Specific quantitative assays are available for a limited number of trichothecenes. Trichothecene analysis can be done by screening methods such as thin layer chromatography (TLC) and ELISA, or analytical methods such as gas chromatography (GC) and high performance liquid chromatography (HPLC). GC instrumentation has been the most frequently used method for experimental work with trichothecenes. Newer methodologies, such as GC-MS and liquid chromatography–mass spectrometry (LC-MS), have an excellent limit of detection (LOD) of 5 ng/g for T-2 toxin in cereals and food, including wheat flour. Improved sensitivity for detection of T-2 and HT-2 toxins in cereal grains by HPLC using different fluorescent labeling reagents with similar LOD has also been recently described. There are no specific therapies for trichothecene toxicoses. Removal from exposure, such as contaminated diet or environment, is essential. For high-level exposure, supportive and symptomatic treatment is indicated. For low-level chronic exposure, cessation of exposure generally results in recovery. Livestock When a history includes decreased feed intake and failure to thrive, mycotoxins should be

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included in the differential diagnosis and feed should be submitted for a mycotoxin screen. Trichothecenes are not found at very high concentrations in tissues, although stomach contents may contain detectable levels if the animals were eating prior to death. Prevention of absorption of trichothecenes from contaminated feed by use of binders, such as clay and zeolitic products, has not been shown to be effective. Research and Chemical Warfare Considerations for T-2 Toxin T-2 toxin is the trichothecene most commonly considered a potential agent of chemical warfare. Neither vaccines nor specific antidotes are readily available. The first step in suspected toxicosis is to stop exposure. If ingestion is the source of contamination, the food source needs to be changed. If exposure is environmental, outer clothing should be removed and exposed skin washed thoroughly with soap and water. Exposed eyes should be treated with copious saline or water irrigation. If exposure has been through ingestion, super-activated charcoal may be used to bind trichothecenes and prevent further absorption. Steroidal anti-inflammatory agents (supportive treatment) and monoclonal antibodies may also be used. Appropriate samples, such as serum, nasopharyngeal swabs, and urine, should be collected from exposed individuals and sent for toxin identification or confirmation using gas chromatography–mass spectrometry (GC-MS) techniques. T-2 toxin is stable in the environment, and resistant to heat and ultraviolet light. Respiratory, skin and eye protection are required for personnel working with trichothecenes. Decontamination of clothing, equipment, and the environment can be performed since T-2 toxin is sensitive to standard household bleach. Other trichothecenes should also be sensitive to this decontamination procedure.

5. ZEARALENONE 5.1. Source/Occurrence Zearalenone is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium, but primarily by Fusarium graminearum

(previously Fusarium roseum). Other fungi include Fusarium culmorum, Fusarium cerealis, Fusarium equiseti, and Fusarium verticillioides. Zearalenone and its metabolites have low acute toxicity but have estrogenic effects following subacute to long-term exposure, with alterations in the reproductive tract, decreased fertility, increased number of resorptions, and decreased litter size. Zearalenone can cause reproductive disorders in farm animals, with prepubertal swine being most susceptible and poultry resistant. Fusarium spp. have a worldwide distribution in temperate and warm climates, infecting corn, wheat, and other grains, and occasionally forages. The highest prevalences of zearalenone are reported in Canada, central and northern Europe, and the United States. Zearalenone occurs in many agricultural products, including cereals, mixed feeds, rice, and corn silage. The most frequently contaminated crop is corn, followed by wheat; barley, sorghum, and rye can also be contaminated. Zearalenone can occur concomitantly with trichothecenes, such as nivalenol and DON, since these compounds are produced by the same Fusarium spp. Interpretation of research on zearalenone toxicity using naturally contaminated feed should be considered in this light. Zearalenone production by Fusarium spp. is favored by wet climates (high rainfall) and especially by wet, cool weather. Zearalenone is primarily a field contaminant but toxin can also be produced during storage, especially when grain has too much moisture when harvested and is not dried properly before storage. Zearalenone is heat stable and therefore can be found in grain-based products such as bread. It has also been found in locally brewed beer in Africa. Alpha-Zearalanol (zeranol, RalgroÒ ), a mammalian metabolite of zearalenone, is used in the USA and Canada as an anabolic agent in beef cattle and sheep. The EU bans it because epidemiological data suggest it can cause central precocious puberty (CPP) in children. It has been proposed as hormonal replacement therapy for postmenopausal women because of antiatherogenic effects observed in animal studies. Zearalenone and its derivatives have been patented for use as oral contraceptives. They can be found in bust-enhancing dietary supplements because they have been associated with breast enlargement in humans.

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5.2. Toxicology Toxin Zearalenone (previously called F-2 toxin) is 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-bresorcylic acid lactone. It is biosynthesized through a polyketide pathway (Figure 39.10). The keto group at C-8 is reduced to a- and b-isomers in mammalian tissues, and these metabolites can also be produced at low concentrations by the fungi. Additional derivatives have been described in corn. Mechanism of Action Zearalenone and its derivatives are the only known mycotoxins whose effects are primarily estrogenic. A three-dimensional model of the zearalenone molecule can be used to demonstrate the similarity of the configuration of this toxin to estradiol. Thus, zearalenone and its metabolites can occupy and activate estrogenic receptors (ER); they have a higher affinity for ER-a than for ER-b. Estrogen-like effects are

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then induced by activation of gene transcription via estrogen-responsive elements. Zearalenone is a strong agonist and a partial antagonist for ER-a, activating both the AF-1 and AF-2 functions of ER-a and inducing the rapid action-mediated response for ER-a in a dose- and cell-specific manner in in vitro studies. The potency of estrogenic effects differs between the stereoisomers, and there are species differences in binding affinity. The potency of transcription and synthesis of estrogen-induced protein in the rat uterus after zearalenone treatment relative to that of 17b-estradiol was 0.07 for a-zearalanol, 0.02 for b-zearalanol, and 0.001 for zearalenone. Zearalenone induces cell cycle progression in the MCF-7 human mammary carcinoma estrogen-dependent cell proliferation assay, with a-zearalenol the most potent inducer of cell proliferation. Relative binding affinity of a-zearalenol to ERs is pig > rat > chicken, which correlates with species sensitivity. Thus, zearalenone and its metabolites are considered mycoestrogens, a subset of naturally

FIGURE 39.10 Chemical structures of zearalenone, a-zearalanol and estradiol. These compounds induce similar responses to estradiol due to binding and stimulation of estrogenic receptors. Zeranol is approved for use as a growth promoter in beef cattle in the United States.

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occurring estrogenic compounds or xenoestrogens, and are classified as endocrine disruptor chemicals (EDCs) (see Endocrine Disruptors, Chapter 37). The uterine and mammary effects are induced by an interaction of zearalenone with estrogenic cytosolic receptors in these organs. Zearalenone also acts on the hypothalamic–hypophysial axis with release of prolactin and luteinizing hormone. Zearalenone has an effect on a number of transcription factors. It can activate the pregnane X receptor (PXR), a human xenobiotic receptor member of ligand-activated nuclear transcription factors. PXR regulates the expression of genes involved in the metabolism of xenobiotics, such as the cytochrome P450 enzymes CP3A4, and has a role in the transcriptional regulation of glutathione-S-transferases, sulfotransferases, and UGTs, as well as a number of transporters. In addition, zearalenone was shown to activate constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR) mRNA levels, as well as a number of CYP enzymes in human hepatocyte cultures. Genotoxicity data for zearalenone are largely negative. It does not cause gene mutations in bacterial test systems, but is clastogenic and aneugenic in vitro and has been confirmed as an in vivo clastogen in mice. DNA adducts induced by high doses of zearalenone in rats and mice are thought to be secondary to oxidative damage since they were reduced by coadministration of the antioxidant a-tocopherol. Oxidative stress has been demonstrated in zearalenone-exposed mice and swine by increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD) and glutathione peroxidase (GSHPx). Zearalenone and its metabolites induced MDA formation and apoptotic cell death in a number of cell culture systems that could be inhibited by antioxidants. Thus oxidative damage may be responsible for non-estrogenic cytotoxicity induced by zearalenone at high doses. Species Susceptibility Zearalenone has little toxicity following administration of single oral or intraperitoneal doses in any species. Effects of oral administration for up to 90 days appear to be primarily dependent on the estrogenic activity of zearalenone and/or its metabolites, with pigs and sheep being more

sensitive than rodents. The pig is the most sensitive, possibly due to greater formation of a-zearalenol relative to other species. The no observed effect level (NOEL), based on estrogenic effects in responsive tissues and reproductive performance, is 40 mg/kg bw per day in pigs as compared to 3 mg/kg bw per day in rats. Based on limited studies, the horse appears to be less sensitive than pigs and ruminants. Poultry do not appear to be affected by zearalenone. Biodistribution, Metabolism, and Excretion Zearalenone is readily and rapidly absorbed from the gastrointestinal tract, with about 85% absorbed in the pig after a single oral dose. Enzymatic reduction results in formation of a-zearalenol (more estrogenic than zearalenone) and b-zearalenol (less estrogenic) as well as smaller amounts of a- and b-zearalanol. Zearalenone is also monohydroxylated by CYP1A2 and, to a lesser extent, CYP3A4, with the major oxidative metabolites being catechols that can undergo oxidation to quinones, which can then redox cycle and covalently modify biological macromolecules such as N-acetylcysteine. Zearalenone and its reduced metabolites undergo phase 2 conjugation with glucuronic acid and sulfate in the intestine, liver, and other organs. There is significant species variation in the extent of zearalenone metabolism, which could explain differences in susceptibility. For example, greater amounts of a-zearalenol, the more estrogenic metabolite, are formed in man and pig compared to rodents. Ruminal metabolism by microbes has been documented in vitro with zearalenone reduced to a-zearalenol and to a lesser degree to b-zearalenol. Based on radiolabeled studies in mice, zearalenone is distributed to estrogen target tissues such as uterus, ovarian follicles, and interstitial cells in the testis as well as adipose tissue. Placental transfer of zearalenone and a-zearalenol has been reported in rats and pigs. Zearalenone and zearalenol (as a combination of free and conjugated forms) are excreted relatively rapidly in feces, urine, and to a small extent in milk. Humans and rabbits excrete the zearalenone metabolites primarily in urine, and elimination from the blood is much slower than in rats, dogs, and monkeys; humans had a significantly higher peak fraction of the dose in plasma. Considerable enterohepatic recycling of

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glucuronidated metabolites occurs in swine and rodents, extending the half-life of plasma zearalenone and prolonging its estrogenic effects. Excretion in swine is primarily through the urine. Zearalenone residues do not persist in animal tissues, although a small amount (less than 1%) of the dose is excreted in milk. Thus, significant contamination of the food chain does not occur through consumption of animal products, with the possible exception of lipophilic metabolites present in egg yolk.

5.3. Manifestation of Toxicity in Animals The predominant toxicologic effect is related to the estrogenic activity of zearalenone and its metabolites resulting in adverse effects on the endocrine organs, male and female reproductive systems, and mammary tissue (see Endocrine System, Chapter 58; Male Reproductive System, Chapter 59; Female Reproductive System, Chapter 60; Mammary Gland, Chapter 61). Toxicity to the liver, hematopoietic system, and immune system (in vitro) has also been described in animals exposed to high doses. Anabolic activity has led to the use of a-zearalanol as a fattening agent in cattle and sheep. In domestic animals, zearalenone can interfere with the estrus cycle, ovulation, conception, and implantation; induce embryonic death; reduce fetal weight and litter size; and impair neonatal survival. Swollen vulvas and vaginal and rectal prolapses are common clinical signs of exposure, especially in the pig, which is the most sensitive species, with the female prepubertal pig the most sensitive. Many outbreaks of naturally occurring mycotoxicosis due to exposure to zearalenone in the diet have been reported in swine and less often in cattle and sheep. Naturally contaminated feed, often used to study zearalenone toxicity, is frequently also contaminated by trichothecenes. Although trichothecenes are not estrogenic, overall interpretation of study results must account for the presence of trichothecenes. Laboratory Animals Zearalenone has a low acute toxicity with oral LD50 values of greater than 2,000 mg/kg bw in mice, rats, and guinea pigs. In female rodents, zearalenone has adverse effects on the reproductive tract, fertility, and embryo survival, but

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teratogenic effects have not been observed. Effects are similar to those described below for swine but occur at much higher doses, in the range of 1–10 mg/kg bw per day. In addition, there were changes in the weights of adrenal, thyroid, and pituitary glands, and in serum levels of progesterone and 17b-estradiol. In males, adverse effects on testosterone synthesis, sexual behavior, accessory sex organ weights, testicular morphology, and spermatogenesis have been observed, but again, at much higher doses than in swine. In rats, germ cell apoptosis was identified at stages I–VI of spermatogenesis 12 hours after dosing. This was proposed as the principal mechanism contributing to germ cell depletion and testicular atrophy following zearalenone exposure. Of particular interest are long-term studies on zearalenone, including subchronic and carcinogenesis bioassays using purified zearalenone conducted by the National Institute of Environmental Health Sciences (NIEHS) National Toxicology Program (NTP) using B6C3F1 mice and Fischer 344/N rats (http://ntp.niehs.nih.gov/ ntp/htdocs/LT_rpts/tr235.pdf). The animals were fed zearalenone at concentrations of up to 3000 mg/kg of diet (450 or 300 mg/kg bw per day in mice or rats respectively) for 13 weeks or at up to 100 mg/kg of diet to mice and 50 mg/kg diet to rats for 103 weeks (carcinogenicity bioassay). In the 13-week study, several high-dose treated female mice died. Most female mice had endometrial hyperplasia that was not dose related. Male mice had atrophy of the seminal vesicles and cytoplasmic vacuolization of the adrenal at  1000 mg/kg diet and squamous metaplasia of the prostate at the 3000 mg/kg diet. Dose-related osteopetrosis was seen at  100 mg/kg and myelofibrosis at  1000 mg/kg in both sexes. Similar changes were found in the seminal vesicles, uteri, and bones of rats. Fibromuscular hyperplasia of the prostate and atrophy of the testis were present at diet levels of 1000 mg/kg and above. In addition, in both sexes there was chromophobe hyperplasia of the pituitary at  1000 mg/kg and ductular hyperplasia of the mammary gland at the highest dose. In the mouse carcinogenicity bioassay, treatment-related non-neoplastic lesions were found only in females. These lesions were estrogenrelated: uterine fibrosis, cystic ducts in the

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mammary gland, and myelofibrosis of the bone marrow. There was a dose-related increase in hepatocellular adenomas in females, and a doserelated increase in pituitary adenomas in both sexes. These tumors are believed to be due to the estrogenic effects of zearalenone. In rats, non-neoplastic treatment-related lesions included inflammation of the prostate, testicular atrophy, hepatocellular cytoplasmic vacuolation in males, and an increased incidence of chronic progressive nephropathy in both sexes. No treatment-related increase in neoplasms was found in these rats or in FDRL Wistar rats in a similar study. Because of the limited evidence of carcinogenicity, the IARC allocated zearalenone to Group 3 (not classifiable as to its carcinogenicity to humans) based on inadequate evidence in humans and limited evidence in experimental animals. A NOEL of 0.1 mg/kg bw per day was derived in rodents based on the absence of increased uterine weight. When administered to dogs, zearalenone reduced the number of corpora lutea and caused uterine hyperplasia in females and arrested spermatogenesis in males. This is of interest since endometrial hyperplasia predisposes to pyometra, a life-threatening bacterial infection of the uterus that is not uncommon in the dog pet population. Experimentally, estrogen priming followed by progesterone stimulation can reproduce the cystic endometrial hyperplasia that precedes pyometra. Swine Swine are the most susceptible species to zearalenone toxicity, with target organs consisting of the ovary, uterus and vulva. Estrogenic effects may occur in swine fed diets containing > 1 ppm zearalenone. In prepubertal gilts, the most sensitive physiological state, the effects include swelling and edema of the vulva (Figure 39.11), vaginal and rectal prolapse, uterine enlargement and edema, atrophy of the ovaries, enlargement of the mammary glands, and a thin catarrhal exudate from the vulva. Experimental exposure of prepubertal gilts to estradiol, zearalenone, and Fusarium graminearum-inoculated corn induces similar histological changes (see Female Reproductive System, Chapter 60). Cervical changes consist of epithelial metaplasia, the normal double layer of columnartype cells replaced by a stratified squamous cellular layer up to 15 cells thick and irregular

FIGURE 39.11 Vulval hypertrophy and edema, estrogenic effect, sow. Zearalenone, a mycotoxin commonly found in corn, has estrogenic effects that result in vulval edema. Photograph courtesy of Dr J. Simon, University of Illinois. Reproduced from Zachary, J. F. (2007) Female reproductive system. In: Pathologic Basis of Veterinary Disease, 4th Ed. (McGavin, M., and Zachary, J.F., eds), Mosby Elsevier, Fig. 18-49, p. 1306, with permission.

in distribution. Similar but more severe changes were seen in the vagina. Interstitial edema, together with cellular proliferation, account for the clinically observed tumefaction (swelling) of the vulva and enlargement of the uterine horns with hypertrophy of all uterine layers. This uterotrophic effect is the basis for the ratuterus bioassay, which has proven to be a practical laboratory method for testing a variety of compounds for their potential estrogenic effects. Ovarian changes are variable, with the only consistent effect being an irregularity in the size of the developing follicles. The mammary gland and nipples enlarge due to interstitial edema and ductal hyperplasia. Reproduction may be affected in sows, but higher levels of exposure are required. The changes induced by zearalenone depend on the time of dose administration in relation to the estrus cycle, as well as on the amount administered. Anestrus or nymphomania may be noted. In sows exhibiting nymphomania, ovaries are atrophic and lack corpora lutea and Graafian

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follicles, indicating follicular atresia. The effects of zearalenone on the uterus, cervix, vagina, and mammary gland in sows are similar to the effects in prepubertal gilts. Reduced litter size due to fetal resorption (mummification) and/or implantation failure occurs when exposure to dietary zearalenone occurs at 7–10 days post-mating. Pigs may be weak or stillborn, and occasionally exhibit swollen vulvas at or shortly after birth (juvenile hyperestrogenism). Gilts may develop pseudopregnancy with multiple persistent corpora lutea in the ovary indicating a luteotrophic property of zearalenone. Uterine changes, characterized by both hyperplasia and hypertrophy, are indicative of estrogenic effects from zearalenone as well as a progesterone effect due to the persistent corpora lutea (see Female Reproductive System, Chapter 60). Squamous metaplasia of the vaginal lining as well as alveolar development and ductular squamous metaplasia are found in the mammary gland. Field observations of zearalenone-induced abortions are thought to be largely erroneous since estrogens are luteotropic in swine. Instead, it is suspected that implantation failure followed by pseudopregnancy leads to a diagnosis of abortion. Zearalenone affects granulosa cell steroidogenesis, oocytes, and fertilized ova, based on in vitro studies. The NOAEL for zearalenone in pubertal female pigs (gilts) is 40 mg/kg bw per day. Prolongation of the luteal phase of the estrus cycle, accompanied by high serum progesterone levels, appears to be the most sensitive adverse effect, with a lowest observed effect level (LOEL) of 200 mg/kg bw per day. In prepubertal gilts, the LOEL for zearalenone varies with the criteria and study;  17.6 mg/kg bw per day based on vulval volume (swelling and lengthening) and > 20 mg/kg bw per day based on histological evaluation of the uterus (cell proliferation and hyperemia). The overall NOEL for zearalenone is 10 mg/kg bw per day. In boars, adverse effects on testosterone concentrations, sexual behavior, testis and secondary sex organ weights, testicular morphology, and spermatogenesis have been observed following exposure to zearalenone or its metabolites. Adverse effects were observed at 20 mg zearalenone/kg or more but not at 1 mg/kg in feed (for 2 months). In prepubertal boars, zearalenone

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may reduce libido and plasma testosterone concentrations. In castrated or prepubertal males, there is also enlargement of mammary glands and swelling of the prepuce. Testicular atrophy has also been reported. Cattle and Sheep Cattle and sheep are much less sensitive than pigs to the estrogenic effects of zearalenone. Zeranol, in the form of an implant, is widely used in the USA as a growth promoter in beef cattle and is also used in sheep. In cattle, zearalenone toxicity may be associated with precocious udder development in heifers and reduced fertility in breeding animals; however, most animals exposed to zearalenone for brief periods of time will recover normal reproductive function. Severe toxicosis may be characterized by ovarian fibrosis and changes in the fallopian tube and uterus which could conceivably have more prolonged effects on reproduction. Vulval swelling, and decreased feed intake and milk production also have been described. Ewe infertility with a decrease in ovulation rate and lambing percentage has been reported.

5.4. Human Risk and Disease EFSA established a TDI for zearalenone and its metabolites of 0.25 mg/kg of body weight in 2011. This TDI is well above the mean dietary intakes for zearalenone in North America, which have been estimated at 20–30 ng/kg bw per day. The main source of exposure is corn (Zea mays) and corn products, although other grains and possibly eggs, which accumulate a zearalenone metabolite in the yolk, also contribute. Meat, including that from cattle implanted with azearalanol, and milk are not considered significant sources of exposure. In Europe, the highest concentrations of zearalenone were found in wheat bran and corn and products thereof. Significant amounts were also found in corngerm and wheat-germ oils. Zearalenone, like most mycotoxins, is heat stable. However, cooking under alkaline conditions and extrusion cooking significantly decreases the concentration. Because of the ability to interact with estrogen receptors, dietary zearalenone and metabolites could increase the estrogen burden in humans (see Endocrine Disruptors, Chapter 37).

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High concentrations of zearalenone identified in foods in cases of estrogen-related adverse effects in humans, such as precocious puberty and breast cancer, led to speculation regarding possible cause and effect. Zearalenone was suggested as a cause of premature thelarche (onset of secondary breast development) and precocious puberty in children in regions of Italy, Hungary, and Puerto Rico, partly based on detection of these compounds in blood and/or food samples. However, insufficient evidence is available to confirm this assertion. A more recent study in healthy girls aged 9–10 years from New Jersey found that girls with detectable urinary zearalenone levels tended to be shorter and less likely to have reached the onset of breast development. The authors postulate an anti-estrogenic effect of zearalenone or its metabolites. Urinary zearalenone levels were associated with beef and popcorn intake. In China, zearalenone was extracted from buckwheat contaminated with Fusarium spp. that was linked to “endemic breast enlargement.” Because zearalenone is thought to be associated with breast enlargement, it is found in many breast-enhancing dietary supplements. The suggested zearalenone link to human breast cancer is based on in vivo studies showing mammary gland enlargement and ductal hyperplasia, and on in vitro studies in which zearalenone stimulated the proliferation of human breast cancer MCF-7 cells containing human ER through regulation of the cell cycle and prevention of apoptosis. However, zearalenone decreased the incidence and multiplicity of 7,12–dimethylbenz(a)anthracene (DMBA)induced mammary tumors when given to prepubertal rats, possibly due to increased differentiation of the mammary epithelial tree.

thin layer chromatography (TLC), or gas chromatography with mass spectrometry (GC-MS); ELISA-based tests can also be used and are readily available from commercial suppliers. Quantification can be achieved with matrix calibration or by using stable isotope-labeled standards. Cleaning and selection steps applied to grains after harvesting lead to a decrease in concentration of zearalenone in grains for food consumption. No US FDA action, advisory or guidance levels have been established for zearalenone in feed. However, the EU has guidance values in feedstuff with a moisture content of 12% of 0.1 mg/ kg or ppm for piglets and gilts, 0.25 mg/kg for sows and fattening pigs, and 0.5 mg/kg for calves, dairy cattle, sheep, and goats. For human consumption, the EU has a maximum limit of 0.35 mg/kg in unprocessed corn and 0.4 mg/kg in refined corn oil. There are no binders recommended as efficacious for zearalenone absorption by the US FDA. In addition to concern over the presence of zearalenone in human and livestock diets, diets prepared for laboratory animals and pets also need consideration. The potential of diets containing zearalenone to confound research on endocrine-disrupting chemicals (EDCs) or evaluation of estrogenic effects is especially problematic. In addition, with the expanded use of pigs and minipigs in research and for pharmaceutical safety evaluation (see The Use of Minipigs in NonClinical Research, Chapter 13), care must be taken to avoid the potential confounding effect of zearalenone in the diet of the most sensitive species.

6. FUMONISINS 6.1. Source/Occurrence

5.5. Diagnosis, Treatment, and Prevention In domestic animals, clinical signs together with the identification of zearalenone in the diet form a strong basis for diagnosis. The diagnosis usually is confirmed when normal reproductive function returns after withdrawal of the contaminated feed, and removal of contaminated feed is generally curative. Zearalenone in foodstuffs can be determined by HPLC coupled to fluorescence detection, triple quadrupole mass spectrometers (MS),

Fumonisins are mycotoxins produced by Fusarium verticillioides (formerly Fusarium moniliforme Sheldon), Fusarium proliferatum, and a few other Fusarium species. Production by other species has also been reported, including Aspergillus niger and at least one Tolypocladium (Tolypocladium inflatum) species. Both the fungi and fumonisins are found worldwide in corn (Zea mays) as well as other cereals. They have on occasion been found in other crops, including cowpeas and asparagus. As an animal and human health

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threat, however, fumonisins are associated with corn and corn-based foods. More than 30 homologs have been identified, and the number continues to grow. Fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) are most common, and are produced in an approximate ratio of 6 : 3 : 1. Fumonisin concentrations vary geographically, often differing between nearby locations. Factors favoring fungal growth and toxin production include heat stress, insect damage, high humidity, and often a delay in harvest, as well as improper (wet) storage. Insects that damage corn, such as the European corn borer, provide a means for the fungus to invade the plant. Resistance to the corn borer, as found in some strains of genetically modified corn (GMO), e.g. transgenic corn expressing Bacillus thuringiensis kurstaki (Btk) toxin, decreases fungal invasion. Accordingly, fumonisin concentrations were found to be lower in GMO corn than in unmodified cultivars that were grown under conditions favoring high amounts of insect damage. While fumonisins occur worldwide, especially high concentrations have been found in China and southern Africa: fumonisin concentrations in “home grown” corn from Linxiang, China and areas of the Transkei, southern Africa exceeding 100 ppm have been occasionally reported. Much lower concentrations of fumonisins, less than 1 to a few ppm, are generally found in corn from the USA and South America, while corn from Canada is virtually free of fumonisins. However, concentrations in excess of 10 ppm are not infrequently found and concentrations exceeding 100 ppm occur given appropriate climactic conditions, such as the 1989 drought in the midwestern USA. Because fumonisin concentrations periodically can be high, there is a continuing possibility of episodic outbreaks of fumonisin-related diseases in susceptible animals such as horses and pigs. Fumonisin concentrations in screenings and cracked, broken, or otherwise damaged kernels are generally higher than in whole grains. However, since the fungus is endophytic, normal-appearing corn may contain fumonisins. Hidden (masked) fumonisins are matrixassociated or matrix-bound forms present in corn or foods that are not detected by routine analytical methods. The contribution of hidden fumonisins to the total fumonisin content

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of corn or food products is not firmly established, although some studies suggest that it might be considerable. The chemical nature of the mycotoxin–corn (or food)–matrix interactions, the prevalence of hidden fumonisins in corn or food products, or their contribution to bioavailability and toxicity are not as yet established.

6.2. Toxicology Toxin Structure Fumonisins have a long chain carbon “backbone” with two tricarballylic acid moieties attached (Figure 39.12). Fumonisins B2, B3, and B4 lack one or more of the hydroxyl groups at the C5 or C10 positions. The primary amino function at C2 is important. Homologs having acetyl-, carboxymethyl-, N-(1-deoxyfructos-1yl)- or other R-groups linked to the nitrogen do not elicit biological activity. Hydrolyzed fumonisins, which lack the tricarballylic acid functions, are produced by base hydrolysis and therefore may be found in alkaline cooked foods such as masa and tortillas. In its purified form, hydrolyzed FB1 elicited negligible to no biological activity in various rodent studies. However, alkaline cooked corn culture materials (corn fermented under experimentally controlled conditions) only containing hydrolyzed fumonisins did elicit the biological activity typical of fumonisins. Partially hydrolyzed forms also have been identified in the feces of rats and nonhuman primates, and may be formed by bacteria in the intestinal tract. Species Susceptibility Fumonisins affect a broad range of species, including horses, swine, sheep, cattle, fish, poultry, non-human primates, mink, rabbits, and rodents. Naturally occurring disease occurs in horses and pigs, with horses being the more susceptible species. Hepatotoxicity is elicited in all and nephrotoxicity in many of the species evaluated to date. In addition, fumonisins cause species-specific syndromes such as equine leukoencephalomalacia (ELEM) and porcine pulmonary edema (PPE) (Table 39.7). Cardiotoxicity is the most recently recognized type of toxicity, and has been reported in pigs and horses. In pigs, cardiotoxicity appears to be the

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FIGURE 39.12 Chemical structure of fumonisin B1 and the sphingoid bases, sphingosine and sphinganine. Because of the structural similarity of the fumonisins to the sphingoid bases, they bind to and inhibit ceramide synthase. Figure reproduced from Voss et al. (2007) Fumonisins: toxicokinetics, mechanism of action and toxicity, Anim. Feed Sci. Technol. 137, Fig. 1, p. 303, with permission.

cause of pulmonary edema, which is usually lethal. Importantly, and in contrast to other Fusarium verticillioides-produced mycotoxins, the toxicity and lesions associated with corn molded with Fusarium verticillioides or Fusarium proliferatum have been experimentally reproduced with purified FB1. Among farm animals, horses and donkeys are most susceptible. The amount of exposure

required to induce ELEM, a disease unique to equidae, has not been established, but is certainly quite low, perhaps less than 5 ppm. Swine are also susceptible, although much more highly contaminated feed is required to induce pulmonary edema in swine than to cause ELEM in horses. Cattle and poultry are much more resistant. Among laboratory species, male SpragueDawley (SD) and Fischer 344 rats are extremely

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TABLE 39.7

Species-Specific Morphologic Effects in Experimentally-Induced Fumonisin Toxicity Species

Organ

Horse

Pig

Cattle (calves)

Sheep (lambs)

Rabbits

Rats

Mice

Non-human primates

Liver

þþ

þ

þ

þ

þ

þ

þ

þ

Kidney

þ/

þ/

þþþ

þþþ

þþþ

þþþ

þ

þ

Brain

þþþ















Heart

*

*











þ

Lung



þþþ













* Cardiotoxicity without lesions þ/, reported in some studies. Table modified from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Table IV, p. 675, with permission.

sensitive to the nephrotoxic effects of fumonisins. Diets containing 9 ppm FB1 elicited renal apoptosis in male SD rats when given for up to 90 days. Rabbits are also quite sensitive to renal effects. Mice exhibit kidney lesions, but only at much higher exposure concentrations. Positive carcinogenicity results have been obtained in three chronic (2 years or longer) bioassays of FB1 in rodents. In each case, kidney or liver tumors were induced when FB1 in the diet was  50 ppm, even though the target organ differed by species, strain, and sex of the animals. Biodistribution, Metabolism, and Excretion Phase 1 and 2 metabolism of (parent or nonhydrolyzed) fumonisins by mammalian or avian species has not been reported. Intestinal microflora appear capable of removing the tricarballylic acid groups, leaving the partially or fully hydrolyzed molecule. Absorption and biodistribution patterns of FB1 (FB2 is mentioned below) in different species are similar. Fumonisins are poorly absorbed from the gut, with estimated gastrointestinal absorption probably 10% or less of the dose. After oral, intraperitoneal, or intravenous administration, FB1 is rapidly cleared from the blood and eliminated in feces, with relatively low amounts excreted in urine. FB1 is excreted in bile with significant enterohepatic recirculation occurring in swine, thus increasing the half-life in that species. Tissue burdens are low, with the highest concentrations occurring in liver and kidneys. FB2 behaves similarly, while

little information on absorption and biodistribution of other fumonisins is currently available. The formation of small but detectable amounts of several N-acyl hydrolyzed FB1 ceramide analogs has been demonstrated in vivo. This metabolic pathway has been investigated only in rats and the biological significance, if any, of N-acyl metabolites has not been determined. Mechanisms of Action FB1 and other fumonisins bear a striking structural similarity to the sphingoid bases, sphinganine and sphingosine (Figure 39.12). Fumonisins bind to and are potent inhibitors of ceramide synthases. These enzymes (six, designated CerS1–CerS6, have been characterized) catalyze ceramide formation from sphinganine (or sphingosine) and palmitate or other long chain fatty acids. Inhibition of ceramide synthase thus disrupts overall sphingolipid metabolism and leads to, among other changes, increases in cellular sphinganine and sphingosine concentrations, increases in the 1-phosphate metabolites of these sphingoid bases, and decreases in cell ceramide and complex sphingolipids. Sphingosine 1-phosphate is a particularly important signaling molecule that exerts diverse physiological effects by acting as a ligand for a family of membrane bound G protein-coupled sphingosine 1-phosphate receptors. Together, ceramide, the sphingoid bases, and sphingosine-1-phosphate regulate a variety of cell functions, including apoptosis and cell replication. Their physiological actions interact with those

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induced by some cytokines, such as tumor necrosis factor alpha (TNF-a) or FAS ligand (CD95-ligand), that also induce apoptosis and otherwise influence cell survival and replication. Apoptosis is the earliest histopathological finding in liver and kidney following fumonisin exposure. Thus, elucidating the relationships between fumonisins, sphingolipids, and apoptosis is important for ultimately understanding the molecular mechanism and mode of action of these mycotoxins. Rodent studies have shown that tissue sphinganine and sphinganine plus sphinganine-1-phosphate concentrations, indicative of ceramide synthase inhibition, increase at doses that are too low to cause apoptosis, while at higher doses increased sphinganine concentrations are positively correlated with increased apoptosis in target organs. As tissue injury and/or dose increases, mitosis and regeneration, cell necrosis (as opposed to apoptosis), and other manifestations of fully developed fumonisin toxicosis become evident. Together, this suggests that the basic sequence of events is inhibition of ceramide synthase; disruption of sphingolipid metabolism and sphingolipid regulatory function; apoptosis; and necrosis, mitosis, and compensatory cell proliferation. Induction of neural tube defects (NTD) by FB1 is discussed in Section 6.3, on manifestations of toxicity in animals. The mechanism(s) of NTD induction in mice by FB1 has not been established but likely involves one or more interrelated cellular processes. There is some evidence that depletion of complex sphingolipids associated with the folate binding protein reduces folate uptake and utilization, which in turn increases the risk of NTDs. Other possibilities include cytotoxicity resulting from oxidative stress and signaling by cytokines or modulation of sphingosine 1-phosphate receptor-linked pathways by elevated levels of circulating sphingosine or sphinganine 1-phosphate. Supporting evidence for the latter includes induction of NTDs in LM/Bc mice by the sphingosine 1-phosphate receptor modulator FTY720. Pathophysiological evidence indicates that cardiac failure due to decreased cardiac contractility is the cause of pulmonary edema in swine. Furthermore, sphingoid bases such as sphingosine or sphingosine-1-phosphate are now being recognized as compounds that influence cardiovascular physiology, theoretically providing

a biochemical mechanism for fumonisin cardiotoxicity. Inhibition of L-type calcium channels in the myocardium and in the vasculature by increased cardiac or serum sphingosine is the currently proposed mechanism of action. In horses with ELEM, sphingolipid alterations in brain tissue were not evident, suggesting that the nervous system is affected secondarily. The primary site of action may be the cardiovascular system, since cardiovascular changes in the horse are similar to those observed in swine. In addition, L-type calcium channels occur in high concentrations in the cerebral arteries, which are responsible for autoregulation of blood flow to the brain, which is of special importance when the horse lowers its head to eat and drink. Other molecular events resulting from fumonisin exposure are poorly characterized. It is generally accepted that fumonisins do not directly interact with DNA, and no fumonisin–DNA adducts have been found. No evidence of genotoxicity was found upon evaluation of FB1 by various means, including the Salmonella typhimurium assay, unscheduled DNA synthesis assays in primary hepatocyte cultures and in rat liver after oral exposure to FB1, SOS chromotests, and differential DNA repair and transforming activity studies in mouse embryo cells in vitro. In contrast, lipid peroxidation was induced in liver in vivo, in hepatocytes in vitro, and in rat liver nuclei in vitro by FB1. Evidence of DNA damage secondary to reactive oxygen species (ROS) or other undefined mechanism(s) has been demonstrated by Comet assay in target tissues of rats fed fumonisins. a-Tocopherol was protective in FB1-treated hepatocytes, a finding that suggests oxidative damage to DNA and other cellular macromolecules may also be important from a mechanistic standpoint. FB1 causes renal and hepatic neoplasia in rodents, as discussed in Section 6.3. The mechanism of carcinogenesis is unknown, but most evidence points to a non-genotoxic mode of action. One theory is that tumors arise as the balance between cell death and regeneration becomes disturbed, or if physiological controls such as those governing mitosis, intercellular communication, and growth inhibition are lost (see Carcinogenesis: Mechanisms and Manifestations, Chapter 5).

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6.3. Manifestations of Toxicity in Animals Horses Onset of naturally occurring disease may occur as early as 7 days after exposure to a contaminated diet, but signs usually are first seen after 14–21 days; occasionally onset may be delayed 90 days or more. Outbreaks that affect several horses on the same farm are common. In 1901–1902, over 2,000 horses died in the USA as a result of ELEM; in 1934–1935, over 5,000 horses died in Illinois alone. In a given outbreak, the overall morbidity is generally low, less than 25%, but mortality usually approaches 100% in affected animals. This disease also occurs in donkeys. Two syndromes are described in horses with naturally occurring disease: the neurotoxic and hepatotoxic forms. These forms may appear independently or concurrently. In the field, high-dose exposure is thought to increase the likelihood of the hepatotoxic form, with the more frequently encountered lower doses favoring the neurotoxic form, ELEM. The clinical course of the neurotoxic syndrome is generally short with an acute onset of signs followed by death within hours or days. Decreased feed intake, depression, ataxia, blindness, and hysteria are reported. Anorexia occurs due to glossopharyngeal paralysis, and paralysis of the lips and tongue, with loss of ability to grasp and chew food. Incoordination, circling, ataxia, head pressing, marked stupor, and hyperesthesia are common, as are hyperexcitability, profuse sweating, mania, and convulsions. Acutely affected animals often progress through the manic and depressive stages of the syndrome within 4–12 hours of onset and become recumbent and moribund. Death may occur without clinical signs. The hepatotoxic syndrome usually takes 5–10 days from time of onset of clinical signs to death. Icterus is usually prominent, and there may be edema of the head and submandibular space, as well as oral petechiae. Elevated serum bilirubin concentration and liver enzyme activities are typically present. Terminal neurologic signs may be noted, possibly due to secondary hepatoencephalopathy. The liver is often small and firm, with an increased lobular pattern. Centrilobular necrosis and moderate to marked periportal fibrosis can be observed histologically. In the classical neurotoxic form, there is liquefactive necrosis of the white matter, primarily in

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the cerebrum (Figure 39.13A). The liquefactive necrosis, most commonly located in the subcortical white matter, is often evident grossly as cavitation or discoloration. Histologically, necrosis with influx of gitter cells, edema, and hemorrhage are primary lesions (Figure 39.13B). Some cases may only exhibit histologic lesions consisting of perivascular edema and hemorrhage, with infiltration of mononuclear and plasma cells and occasionally eosinophils. Experimental administration of purified FB1, either per os or iv, can induce both neurologic and hepatic disease, with these generally occurring concurrently. The clinical signs and time course of the neurologic disease are similar to those in naturally occurring disease. Horses with neurologic disease had increased protein in the cerebrospinal fluid and other changes consistent with vasogenic cerebral edema. Histologic lesions characterized by perivascular edema and hemorrhage, primarily of the white matter, were found in both brain and spinal cord. Serum biochemical evidence of hepatic injury was present in horses with neurologic disease as well as in those horses given a dose that did not induce neurologic disease. This dose was 0.01 mg/kg for 28 days and approximated oral ingestion of FB1 at 8 ppm. These findings contradict the clinical literature, which suggests that the neurologic form occurs at lower exposure levels while the hepatic form occurs at higher exposure levels. Hepatic lesions were characterized by hepatocellular apoptosis and necrosis. Sphingosine and sphinganine levels were elevated in serum and tissues such as heart, liver, and kidney, but not in brain. An increase in serum cholesterol concentration was also present. Cardiovascular abnormalities in horses with neurologic disease were similar to those described for swine below, and included decreased cardiac contractility, heart rate, arterial pulse pressure, and increased systemic vascular resistance. Swine Outbreaks of a fatal disease in swine fed Fusarium verticillioides-contaminated corn screenings from the 1989 corn crop in the Midwest and Southeast regions of the United States led to the identification of FB1 as the causative agent of porcine pulmonary edema (PPE). Thousands of pigs died in these outbreaks. In Hungary,

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FIGURE 39.13 (A) Leukoencephalomalacia in the left cerebrum of a horse whose diet was contaminated with fumonisins. Focal malacia (necrosis, arrows) is confined to the subcortical white matter. (B) Leukoencephalomalacia in the cerebrum of a horse. At low magnification, the white matter is severely disrupted (coagulated [liquefactive necrosis]) and there is accumulation of proteinaceous fluid, scattered neutrophils, and abundant macrophages. The interface between the gray (G) and affected white matter contains diffuse edema, perivascular hemorrhage (not shown here), and blood vessels with small leukocytic cuffs. Blood vessel walls are degenerate or necrotic, and some are infiltrated with neutrophils, plasma cells, and eosinophils. Inset: Note the influx of blood monocytes that mature into tissue macrophages and become “gitter” cells as they phagocytose necrotic debris. H&E stain. Figures reproduced from Fundamentals of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), Figs 13.20 and 21, p. 403, with permission.

outbreaks of this disease have been reported since the 1950s. A decline in feed consumption is usually the first sign following fumonisin exposure. If fumonisin consumption is high, acute pulmonary edema and, generally, death follows within 4–7 days after onset of feeding the contaminated diet. Typically, deaths cease within 48 hours after withdrawal from contaminated food. PPE has been reproduced experimentally in swine by feeding naturally contaminated feed, FB containing culture material, and purified FB1. Pulmonary edema is not a finding in any other species. Severe pulmonary edema and hydrothorax occur in the acute form of fumonisin intoxication (Figure 39.14A). Edema appears to originate in the interstitium, with perivascular edema and markedly dilated lymphatics a prominent feature early in the disease (Figure 39.14B). Liver injury is similar to that found in other species, and is characterized by scattered hepatocellular apoptosis, necrosis, and proliferation. Pancreatic necrosis has been reported in some studies. Alterations in clinical pathology reflect hepatic injury, and total serum cholesterol concentration is elevated. Progressive and marked elevations in sphinganine and sphingosine are found in serum and in major organs such as kidney, liver, lung, and heart, indicating a major disruption in sphingolipid biosynthesis. Although altered myocardial morphology has not been documented, FB1 decreases cardiac contractility, mean systemic arterial pressure, heart rate, and cardiac output, and increases mean pulmonary artery pressure and pulmonary artery wedge pressure. Therefore, fumonisin-induced pulmonary edema results from acute left-sided heart failure. These changes are compatible with the inhibition of Ltype calcium channels due to increased sphingosine and/or sphinganine. At lower doses, slowly progressive liver disease may occur. Subacute hepatic injury is characterized by hepatocellular cytomegaly, disorganized hepatic cords, and early perilobular fibrosis and chronic injury by icterus with severe hepatic fibrosis and nodular hyperplasia. Additional findings reported in some studies include esophageal plaques and right ventricular hypertrophy due to pulmonary hypertension. As with other mycotoxins, fumonisins appear to affect the immune system. Effects on both specific and non-specific immunity have been

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reported. FB1 decreased phagocytosis and inhibited sphingolipid biosynthesis in swine pulmonary macrophages, and decreased clearance of particulates and bacteria from the pulmonary circulation. Laboratory Animals RODENTS

FIGURE 39.14 Lung from a field case of fumonisin toxicoses, pig. (A) Pulmonary edema: interlobular septa are widely distended by edema. (B) Prominent proteinaceous interstitial edema is present in the pleura (top) and interlobular septum. Note the widely dilated lymphatics (L) present in the septum and pleura (H&E, 4X) (Images courtesy of Sandeep Akare). Figure 39.14A reproduced from Fundamentals of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), Fig.e 6.18A, p. 121, with permission. Figure 39.14B reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 14A, p. 679, with permission.

The main target organs in rodents and rabbits are liver and kidney. The pathology has been variously described as “hepatopathy” or “hepatosis,” and “nephropathy” or “nephrosis,” and is similar in animals given purified fumonisin B1, culture material (corn that is infected with a single fungal isolate and then molded under controlled laboratory conditions), or corn naturally contaminated with fumonisins. In the liver, the earliest finding is hepatocellular apoptosis. Affected cells are scattered throughout the hepatic lobules. Inflammation is usually absent. With increasing tissue injury, apoptotic cells become more numerous, and cytoplasmic vacuolation, mitotic figures, and hepatocytes undergoing necrosis are increasingly found. Cytomegaly, anisocytosis, and anisonucleosis occur as injury progresses (Figure 39.15A). Chronic non-neoplastic lesions consist of bile duct and oval cell proliferation, fibrosis, nodular regeneration (regenerative hyperplasia), foci of cellular alteration, and cholangiomatous lesions (Figure 39.15B). There is loss of parenchyma around and between central veins. In mice, Kupffer cells or macrophages containing pigment may be present in the centrilobular zone. Inflammation remains minimal and, when present, is usually associated with focal necrosis. g-Glutamyl transferase (GGT) and glutathioneS-transferase p (GST-P) positive foci are readily demonstrated in rats. Serum biochemical profiles support the microscopic findings. Alanine and aspartate aminotransferase, alkaline phosphatase, and GGT activities, as well as bile acids and bilirubin concentrations, are increased. Hypercholesterolemia occurs readily. Leukocytosis and altered differential leukocyte counts, thrombocytopenia, changes in serum immunoglobulin levels, and other evidence suggest that fumonisins exert subtle immunological or hematological effects. Neoplastic hepatic lesions have been found only in male BD IX rats and female B6C3F1

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FIGURE 39.16 Kidney, subchronic dietary exposure to fumonisin B1 and other fumonisins provided by corn culture material, male rat. Characteristic basophilia of tubules in the outer stripe of the outer medulla (OSOM) and detached apoptotic or preapoptotic cells (arrows). H&E stain.

FIGURE 39.15 Liver, chronic dietary fumonisin B1, mouse. H&E stain. (A) Polyploid hepatocytes (arrows) are characterized by an enlarged hyperchromatic nucleus. Megalocytosis (arrowhead) with nuclear membrane invagination (pseudo inclusion) and oval cell proliferation are also present. (B) Large numbers of oval cells, small basophilic cells (arrowheads), extend out from the periportal region separating hepatocytes. An occasional apoptotic cell is present (arrow). Slide courtesy of Dr D. Caldwell, Health Canada. Figures 39.15A and B reproduced from Fundamentals of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2010), Figs 9.17 (p. 221) 9.21 (p. 223), with permission.

mice. They have been variously classified as neoplastic nodules, hepatocellular adenomas, or carcinomas. Proliferative biliary lesions, such as cholangiofibrosis, angiofibrosis, and cholangiocarcinoma, occur only in rats. Hepatocellular carcinomas in rats range from well-differentiated to more anaplastic, poorly differentiated types. In

female B6C3F1 mice, hepatic tumors ranged from discrete adenomas containing well-differentiated cells to hepatocellular carcinomas with poorly differentiated, anaplastic cells organized in the trabecular or adenoidal patterns commonly found in murine liver carcinomas. The kidney is the other major target organ in rodents and rabbits. In Sprague-Dawley and Fisher 344 rats, males are more sensitive to nephrotoxic effects than females. Rabbits are also sensitive, and display well-developed lesions at relatively low doses. In contrast, mice are relatively resistant to fumonisin-induced nephrotoxicity. Lesions, when found, generally consist of a few scattered apoptotic tubular epithelial cells. As in liver, apoptosis is the earliest finding in the kidney (Figure 39.16). Apoptotic epithelial cells are scattered throughout the proximal convoluted tubules of the outer stripe of the outer medulla. Apoptotic cells detach from adjacent cells and slough into the tubular lumen. Accelerated apoptosis has been defined as a pivotal feature in the renal toxicity of fumonisin (see Kidney, Chapter 47). More advanced lesions extend into the adjacent inner cortex and have both regenerative and atrophic components. Regenerative tubules are lined by epithelium that is basophilic and hyperplastic,

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often cuboidal rather than columnar, and with occasional mitoses. Mitoses are found more readily in advanced cases. Other tubules are atrophic and have flattened, squamous epithelia that make the lumina appear distended, and the basement membrane of affected tubules may be amorphously thickened. Interstitial fibrosis is present in the most advanced cases. Inflammation is not a prominent feature. Decreased kidney weight and clinical signs of tubular dysfunction accompany the renal lesions. The latter includes increased serum creatinine and decreased serum total carbon dioxide. Urine output and water consumption may be transiently increased. Other urinary findings include decreased osmolality; increased enzyme activities of GGT, N-acetyl-b-glucosaminidase, and lactate dehydrogenase; inhibition of r-aminohippurate and tetraethylammonium transport; and proteinuria. Glomerular involvement is negligible. Renal tubular adenomas and carcinomas have been found in F344 male rats fed high doses of fumonisins over an extended period ( 50 ppm for 2 years). Tumor morphology ranged from a clear cell type to a sarcomatous variant composed of spindle-shaped cells. Many of the carcinomas displayed a high degree of anaplasia, numerous mitosis, aggressive infiltration of the surrounding tissue, and metastases to the lung and lymph nodes. The neoplasms arose in kidneys that displayed varying degrees of apoptosis, tubular basophilia, regeneration and hyperplasia, and focal tubular atrophy, suggesting that an imbalance between cell loss and replication played a role in tumor induction. NON-HUMAN PRIMATES

There are no reports on the pathological effects of pure fumonisins in non-human primates. However, two of three baboons fed a diet contaminated with a strain of Fusarium verticillioides, now known to produce fumonisins, died of acute congestive heart failure after 143 to 248 days of exposure. After 720 days of exposure, the third baboon was found to have cirrhosis. Other findings in non-human primates (vervet monkeys) suggesting that fumonisins may have cardiovascular, in this case atherogenic, effects are based on serum chemical findings and include elevated plasma cholesterol, low density lipoprotein C, and apoprotein B, as

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well as fibrinogen and coagulation factor VIII activation. South African scientists have conducted a 13.5year study on the effects of fumonisin-containing diets (varying concentrations) in vervet monkeys. Clinical findings suggestive of liver dysfunction included increased alanine and aspartate aminotransaminase, GGT, and lactate dehydrogenase activities. As in other species, serum cholesterol concentrations were also elevated. Serum creatinine concentrations were increased and creatinine clearance was decreased, suggesting that fumonisins affected renal function. Descriptions of microscopic lesions were confined to the liver, which was clearly a target organ. Lesions were consistent with those seen in other species, and included apoptosis, bile duct proliferation, nodular hyperplasia, and perilobular fibrosis. Mitotic figures and apoptotic cells were present in the nodular structures.

6.4. Human Risk and Disease The risks to humans posed by fumonisins are undetermined at present; however, the potential risk is to populations that consume large amounts of corn on a regular basis. Correlations between consumption of moldy, “home-grown” corn as a dietary staple and high rates of esophageal cancer in the Transkei, southern Africa, and Linxiang, north central China, have been reported. Esophageal cancer in these regions occurs in both men and women, most often at age 50–60 years. It is usually detected late and prognosis is poor. Retrosternal pain and dysphagia are common clinical signs due to the mass causing esophageal stricture. The tumors are located most frequently in the middle third of the esophagus, followed by the lower third and then the upper third. Ulceration is common. The tumors most often are keratinizing, squamous cell carcinomas. A variety of esophageal cytological changes, including dysplasia, chronic inflammation, basal cell hyperplasia, and hyperkeratosis, have been demonstrated in patients without frank malignancy. In southern Africa, esophageal cancer tends to occur in clusters, leading to the formation of “high cancer” and “low cancer” areas, such as seen in the Transkei. Home-grown corn from “high cancer areas” is more frequently contaminated with Fusarium verticillioides and has higher

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average fumonisin concentration than adjacent, “low esophageal cancer areas.” Surveys of corn in China have shown either a higher average fumonisin concentrations or a higher percentage of contaminated corn in “high cancer areas” compared to low cancer areas. It has been speculated that fumonisins also may be an etiological factor for liver cancer in China, especially in regions where fumonisins and aflatoxin occur together. Liver cancer is also one of the more frequently encountered cancers in men from the Transkei. These observations do not conclusively show a link between fumonisins and cancer in man, as a number of other factors have been cited as being the cause of, or contributing to, esophageal and other cancers that are found in these regions. Among these are nitrosamines from food and tobacco, mineral deficiencies in the soil, and poor nutrition. Furthermore, it remains a possibility that other mycotoxins, acting alone or in concert with fumonisins, are the cause of the esophageal cancer that has been associated with Fusarium verticillioides. Support for this possibility comes from the lack of data from chronic experimental studies in rodents and non-human primates that would implicate the esophagus as a target organ of fumonisins. Perhaps of concern for humans is the link between cardiovascular disease and fumonisins documented in swine, horses, and non-human primates, and the consistent finding of elevated cholesterol levels in all species exposed to fumonisins. NTDs are anomalies resulting from failure of the neural tube to fully close during early pregnancy (see Embryo and Fetus, Chapter 62). NTDs present in multiple forms, including spina bifida, exencephaly, anencephaly, and craniorachischisis (externalization of the brain and spinal cord): severe forms are incompatible with life. Fumonisins have been implicated in an NTD outbreak that occurred within the MexicanAmerican population of South Texas during 1990–1991. Implicating factors include higher than average fumonisin concentrations in the 1989 corn crop, a simultaneous increase in Texas ELEM cases (see above), and reports that NTD rates are high in areas of northern China, Guatemala, and southern Africa, where the local population is heavily dependent on corn as a food source.

Retrospective case-controlled investigations conducted in the affected area (1995–2000) indicated that the risk of an NTD-affected pregnancy increased with increasing tortilla consumption during the first trimester of pregnancy up to a critical amount, and decreased thereafter. The same “inverted U”-shaped response was found when NTD outcome and maternal postpartum serum sphinganine/sphingosine ratios, an indicator of fumonisin exposure, were considered. These results, while not definitive, provide the basis for further investigations on fumonisins as potential risk factors for NTDs in humans. Human exposure is dietary, with the toxins found in a variety of commercial foods, including corn meal, corn flour, grits, masa, polenta, snack foods, and beer. In general, corn meal and baking mixes have higher levels than more highly processed products. Sweetcorn and popcorn have low fumonisin concentrations, and residues in meat, milk, and eggs are negligible. Milling and distilling do not appreciably degrade these mycotoxins. Fumonisins distribute unevenly in milling fractions, with higher amounts found in bran, gluten, distiller’s grains, steep liquids, flour, meal, and grits. Cornstarch, an important commercial product used in sweeteners, contains negligible amounts of fumonisins. The effect of cooking on fumonisins is the subject of ongoing research. Some reports suggest that fumonisins are resistant to thermal degradation at temperatures normally used for cooking and baking; however, others indicate that frying or autoclaving reduces fumonisin concentrations. Alkaline cooking (known as nixtamalization) effectively reduces fumonisins in tortillas and other masa products. Reduction is achieved by a combination of extraction into the (discarded) cooking liquid, conversion of fumonisins to the less toxic hydrolyzed forms, and perhaps formation of other reaction products. Depending on the specific conditions, extrusion cooking, which combines high heat and pressure, can significantly reduce fumonisins in the cooked corn products. The amount of reduction is enhanced if glucose or other reducing sugar is added before processing. Extruded corn products and the fumonisin glucose reaction products N-(1-deoxy-D-fructos1-yl) fumonisin B1 and N-carboxymethyl fumonisin B1 exhibited reduced toxicity in bioassay experiments.

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Efforts to develop robust and reliable methods for routinely assessing fumonisin exposures in humans continue. The increase in serum, whole blood or tissue sphinganine or sphinganine 1phosphate concentrations or sphinganine:sphingosine ratios that arise as a result of ceramide synthase inhibition have proven to be useful exposure biomarkers in the laboratory. Their use in epidemiological surveys has, however, been limited, and is problematic due in part to the rapid reversibility of these effects. Methods for measuring fumonisins in urine or tissues have recently been developed. While the use of urinary fumonisin concentration as an exposure biomarker has been limited, this approach shows promise, as illustrated by the results of a study of South African subsistence farmers in which a positive correlation was found between urinary FB1 excretion (normalized to creatinine) and dietary exposure.

6.5. Diagnosis, Treatment, and Prevention In horses and pigs, diagnosis of ELEM or PPE is based on a history of ingestion of corn, particularly corn screenings or unscreened corn, together with characteristic clinical signs and lesions. Detection of approximately 10 ppm fumonisin in horse feed or > 50 ppm in swine feed is highly suggestive of toxicosis. However, lower levels or lack of detectable fumonisin does not eliminate the possibility of fumonisin toxicosis, since feed originally present may no longer be available for testing or false negative results are obtained because mycotoxin distribution in the suspect feed lot is heterogenous. Assays (HPLC derivatization) are now available for fumonisin at most veterinary diagnostic laboratories, and ELISAbased screening tests for fumonisin are commercially available. In addition, elevated sphinganine in serum and/or tissues (frozen or formalin fixed) is an excellent indication that exposure has occurred, although this assay is not routinely available in diagnostic laboratories. There is no known effective therapy. The US FDA recommends that corn and corn by-products intended for equidae and rabbits contain < 5 ppm total fumonisins (FB1, FB2, FB3) and constitute < 20% of the diet, on dry weight basis; for swine and catfish, < 20 ppm total fumonisins (no more than 50% of diet); for ruminants, poultry, and mink, 30–100 ppm

total fumonisins (no more than 50% of diet) depending on age and use; and for pets, < 10 ppm (no more than 50% of diet). A group TDI for fumonisins B1, B2 and B3, alone or in combination, of 2 mg/kg body weight has been set by JECFA. In addition, the US FDA has established advisory levels for total fumonisins (FB1 þ FB2 þ FB3) in corn for human consumption, which varies from 2 to 4 ppm, depending on intended use of the corn. The EU and others have also set various limits for fumonisins in foods.

7. ERGOT ALKALOIDS 7.1. Source/Occurrence Ergot alkaloids are tryptophan-derived mycotoxins produced by various fungi, in particular those that parasitize the seed heads of grasses and small grains (Claviceps spp.) and the endophyte Neotyphodium coenophialum, which infects grasses such as tall fescue. The term “ergot” is used as the common name for the fungus Claviceps spp., which invades the ovary of grasses and cereals, and in reference to the sclerotium, the hard purple or black mass consisting largely of mycelial hyphae that replaces the seed head of grasses and contains ergot alkaloids (Figure 39.17). Ergotism or ergot toxicosis refers to poisoning resulting from ingestion of Claviceps spp. infected grains and grasses or, occasionally, to toxicity following human or veterinary medical use of ergot alkaloids. Livestock disease from ingestion of tall fescue infected with Neotyphodium coenophialum and containing ergot alkaloids is termed fescue toxicosis. Livestock production loss in the USA from fescue toxicosis is estimated at more than $1 billion per year. Since ergot and fescue toxicoses are clinically indistinguishable in species predisposed to both, a more appropriate terminology is ergot alkaloid toxicosis. The more specific term of ergopeptine alkaloid toxicosis is sometimes used for toxicosis due to endophyte-infected tall fescue. Clinical syndromes of ergot alkaloid toxicosis are gangrenous, hyperthermic, reproductive, neurologic (convulsive), and enteric. Vasoconstriction contributes to several of these syndromes, especially to development of

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FIGURE 39.17 grain.

Claviceps purpurea sclerotium on cereal

gangrene and hyperthermia. Clinical syndromes depend to some extent on species affected, with modifying factors including specific ergot alkaloids consumed, amount consumed, length of exposure, age and reproductive status of the animal, animal genetics, ambient temperature, and nutrition. The use of ergot in human medicine officially began early in the 19th century as a means to hasten childbirth. However, the dangers of using ergot in childbirth, including stillbirth, were rapidly recognized and it was recommended that ergot only be used to control postpartum hemorrhage. Current medical uses of ergot alkaloids include ergotamine for migraine headaches, ergometrine to induce uterine contractions, and bromocriptine (synthetic) for hyperprolactinemia-associated dysfunctions, acromegaly, and Parkinson’s disease. Claviceps spp. Members of the genus Claviceps infect more than 600 plant species, including sedges, rushes,

and grasses. Important host plants for Claviceps purpurea include rye, triticale, barley, oats, wheat, millet, Kentucky bluegrass, and brome, orchard, timothy, and quack grasses. In terms of food safety, infection of grains with Claviceps purpurea is of greatest concern, although Claviceps africana found in sorghum and Claviceps fusiformis in pearl millet also need to be considered. Claviceps cyperi, found on the weed yellow nut sedge in South Africa, has caused ergot alkaloid toxicosis in dairy cattle. Claviceps paspali grows on grasses of the Paspalum spp. and causes neurotoxicity in animals manifest as ataxia or “staggers.” This neurologic syndrome is due to indole-diterpenoid tremorgens (paspalitrems) and not due to ergot alkaloids that occur at very low levels. The severity of infection by Claviceps spp. is of high seasonal variability because the extent of infection depends on many factors, including temperature, humidity, and farming practices. Ergot poisoning in humans, sometimes of epidemic proportions, was first described in the Middle Ages, and occurred due to the ingestion of bread made from rye contaminated by Claviceps spp. It was characterized by gangrene and intense burning of the extremities, giving rise to the name “St Anthony’s fire.” Behavioral abnormalities, convulsions, and miscarriages also were reported. Neotyphodium spp Tall fescue (Lolium arundinaceum) is the major plant host for the endophytic fungus Neotyphodium coenophialum, previously known as Acremonium coenophianum, and before that as Epichloe typhina. This organism is a Clavicipitaceae fungus that is transmitted in the seed (not via spores) and can produce ergot alkaloids, primarily ergovaline, as well as other mycotoxins such as loline alkaloids and peramine. Infection with Neotyphodium coenophialum confers drought and stress tolerance, and pest resistance, to the plant. Tall fescue is a major forage grass, grown on an estimated 35 million acres in the United States as well as extensively in Australia and New Zealand. The grass is sometimes allowed to “accumulate or stockpile” for winter grazing, and is cultivated as hay. In the United States, fescue is especially important in the Pacific Northwest, Kentucky, and the Southeast. It is estimated that over 90% of tall fescue fields in the United

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States are infected with this endophyte. Fescue toxicosis has been reported in the United States, Argentina, Australia, Italy, and New Zealand. The toxicoses can manifest as vascular and thermoregulatory disturbances culminating either in local hypothermia (fescue foot) or in generalized hyperthermia (summer fescue toxicosis). In addition, reproductive problems, agalactia (failure of milk secretion), altered lipid metabolism with fat necrosis (lipomatosis), and increased oxidative stress have been reported. Neotyphodium lolii infects many cultivars of perennial ryegrass (Lolium perenne) grown for forage and turf in the United States, Australia, and New Zealand. This endophyte produces a range of tremorgenic indole terpene alkaloids, especially lolitrem B, as well as small amounts of ergot alkaloids such as ergovaline. Ruminants

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and horses ingesting infected ryegrass develop perennial rye grass staggers due to the tremorgens (see Selected Poisonous Plants Affecting Animal and Human Health, Chapter 40).

7.2. Toxicology Toxins There are over 50 ergot alkaloids, and most have a tetracyclic ergoline ring system (Figure 39.18). They can be divided into four major groups based on the substitution at C-8: the clavine alkaloids and 6, 7-secoergolenes, simple lysergic acid derivatives, ergopeptine alkaloids (cyclol ergot alkaloids), and ergopeptam alkaloids (lactam ergot alkaloids). Members of the clavine alkaloids are derived from precursors

FIGURE 39.18 Chemical structure of selected ergot alkaloids and dopamine. Most ergot alkaloids have a tetracyclic ergoline ring system. The structural similarity of ergot alkaloids to dopamine influences action at the dopamine receptor.

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of lysergic acid, e.g., agroclavine. The simple lysergic acid derivatives are amides of lysergic or paspalic acids. Ergopeptine alkaloids (ergopeptides, such as ergotamine) are composed of lysergic acid and a tripeptide moiety that is condensed to a tricyclic system. Ergopeptams are similar to ergopeptine alkaloids. The ergopeptine alkaloids are responsible for the majority of ergot alkaloid toxicoses. The toxic effects of Claviceps spp. infected grains and grasses are due to the ergot alkaloids present in the sclerotia. The predominant alkaloids produced by Claviceps purpurea are ergotamine, ergosine, ergocristine, a- and b-ergocryptine, ergometrine, and ergocornine, and their corresponding epimers (-inine forms; e.g., ergosinine, ergocristinine). Claviceps africana produces primarily dihydroergosine and Claviceps fusiformis primarily agroclavine. The alkaloid profiles and total amounts biosynthesized are highly variable and depend on location, climatic conditions, host plant, and fungal genotype. Ergovaline accounts for about 90% of the ergopeptine alkaloids in endophyte-infected fescue. Therefore, it is a good biomarker of potential toxicity. Fescue toxicosis has been reproduced with ergovaline, ergotamine, and bromocriptine, all ergopeptine alkaloids. Other ergot alkaloids include ergosine, ergonine, and lysergic acid amide (ergine). Lysergic acid has sedative properties as well as effects on the autonomic nervous system (ANS) such as hypersalivation, emesis, dizziness, and diarrhea. The loline alkaloids are saturated pyrrolizidine compounds. Their role, if any, in fescue toxicoses is considered minor. Biodistribution, Metabolism, and Excretion Toxicokinetic data are sparse and mainly limited to ergot alkaloids used for medical purposes. Absorption of orally ingested ergopeptide alkaloids appears limited to the small intestine except for ruminants, where significant absorption can also take place in the rumen. Ruminal microbes release ergot alkaloids from plant material and have the ability to metabolize the released alkaloids. Gastrointestinal absorption of ergot alkaloids is extensive in ruminants, but less so in rhesus monkeys (Macaca mulatta) and rats. Following absorption, ergot alkaloids are rapidly distributed in the body.

Following oral ingestion, more than 90% of absorbed ergotamine is estimated to undergo presystemic metabolism in humans. Oxidative biotransformation in the liver is mediated primarily by the CYP3A4 subfamily so that ergotamine is converted to more hydrophilic metabolites by sequential hydroxylation reactions. Some ergot alkaloids are conjugated with glucuronic acid for biliary excretion. When used therapeutically, bioavailability of ergotamine depends on the route of administration, but can also vary widely between patients or even when given repeatedly to the same patient. Bioavailability is < 5% with oral formulations with peak plasma levels occurring at 1–3 hours. Absorption is higher when ergotamine is administered rectally as suppositories. Lysergic acid diethylamide (LSD) is rapidly absorbed after oral administration and widely distributed to the tissues. LSD crosses the blood–barrier easily and enters the brain, its target site of action. Excretion occurs in urine, bile, and feces, with biliary excretion being the primary elimination pathway for most species whereas urine is the primary route of excretion in ruminants; the horse falls in between. Some ergot alkaloids are excreted to a small extent in milk. Although ergot alkaloids disappear rapidly from blood and tissue, their physiologic effects persist for a long time. For example, ergotamine produces vasoconstriction that lasts for at least 24 hours despite a plasma half-life of about 2 hours. The half-life of ergovaline in sheep following iv administration is estimated as 24 minutes and in horses as 57 minutes. Residues are not found in edible tissues or milk of livestock, although, in one study, low levels were found in subcutaneous fat. Mechanism of Action The wide spectrum of pharmacological activity of ergot alkaloids is attributed to their agonist, partial agonist, or antagonist action on a number of neurotransmitter receptors, especially the adrenergic, dopaminergic, and serotonergic receptors. For example, the marked effects of ergotamine on the cardiovascular system are due to simultaneous peripheral vasoconstriction, depression of vasomotor centers, and peripheral adrenergic blockade. The spectrum of effects depends on the specific alkaloid or combination, species, tissue, and experimental

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or physiologic conditions. However, the primary effects of ergot alkaloids are due to actions on the CNS (central/neurohormonal) and direct stimulation of smooth muscle (peripheral) resulting in vasoconstriction and uterine contraction. Because of their neurohormonal effects, ergot alkaloids are considered endocrine disruptors (see Endocrine Disruptors, Chapter 37). The central neurohormonal effects of ergot alkaloids are due to the structural similarity of ergot alkaloids to biogenic amines. Ergot alkaloids are D1 dopaminergic antagonists. Since these receptors appear to play a role in vasodilation, antagonistic effects may play a role in vasoconstriction that results in gangrene of the extremities. Chronic vasoconstrictive action leads to proliferative lesions of smooth muscle cells in small muscular arteries and arterioles. Ergot alkaloids are D2 dopaminergic agonists with ergopeptine alkaloids like ergovaline having a much higher affinity for binding than the ergoline alkaloids. Agonist-mediated effects on receptors in pituitary lactotrophs result in a reduction of prolactin secretion by the anterior pituitary, which can subsequently interfere with normal mammary gland growth (i.e., lactogenesis) and reduce milk secretion. Because of prolactin’s role in maintenance of luteal function and gonadotropin release, low prolactin levels can also cause an imbalance in reproductive hormones leading to early pregnancy problems in cattle and late pregnancy problems in horses. Prolactin also has a role in lipogenesis, regulation of the hair cycle, and immune function. Altered dopamine and/or prolactin levels also affect the thermoregulatory center in the hypothalamus, resulting in dysregulation that contributes to development of hyperthermia or hypothermia. Some clinical syndromes can be precipitated when ambient temperature falls outside the “normal” range. Cattle on endophyte-infected tall fescue tend to develop the gangrenous syndrome (fescue foot) when the temperature falls below 8
C (46
F), or the hyperthermic syndrome above 31
C (88
F). Laboratory animal studies support the role of heat stress in the hyperthermic syndrome. Other neuroendocrine compounds whose secretion is affected by ergot alkaloids include epinephrine, norepinephrine, melatonin (secreted by the pineal gland), and serotonin. The resulting neurotransmitter imbalances affect growth,

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reproduction, and the ability to respond to seasonal changes in photoperiod and environmental temperature. Ergot alkaloids are a1-adrenergic receptor antagonists and a2-adrenergic agonists. One of the important effects of a2-adrenergic agonist activity is vasoconstriction that can lead to elevated blood pressure and damage to the capillary endothelium (see Cardiac, Vascular, and Skeletal Muscle Systems, Chapter 46). Agonist action on serotonergic2 receptors by ergot alkaloids can also contribute to vasoconstriction, especially in uterine and umbilical arteries as shown in vitro. Ergot alkaloid mediated contraction of smooth muscle, both vascular and non-vascular, occurs independently of innervation or other chemical mediators. The blood vessels in most vascular beds are constricted by the ergot alkaloids, but the arterioles are most sensitive. The resulting vascular occlusion can be demonstrated by arteriography and leads to ischemia of the tissues perfused by the affected blood vessels. The ultimate outcome is variable: the tissue may become gangrenous if ischemia is persistent, or, conversely, it is possible for blood flow to be reestablished without permanent tissue damage. The limbs, toes, ears, and tail are most often affected, but usually not all in the same individual or animal. Occasionally, scattered areas of the skin also may become gangrenous. Vasoconstriction of blood vessels in the skin decreases the ability to dissipate heat and can contribute to hyperthermia. When exposure occurs during the third trimester of pregnancy, ergot alkaloids have an oxytocin-like (oxytocic) effect on the uterus and can induce labor since uterine muscle is more sensitive to ergot alkaloids at this time than other smooth muscle. In early pregnancy, these alkaloids stimulate the cervix more than the uterus. Other a2-adrenergic agonist effects reported include decreased liver enzyme concentrations in the blood, oxidative stress, a decrease in release of antidiuretic hormone from the pituitary, and decreased production of aldosterone in the adrenal. The decrease in antidiuretic hormone and aldosterone results in excessive urination. Serotonergic agonism can also affect the thermoregulatory center in the hypothalamus and can cause appetite suppression.

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Hallucinogenic effects can be produced by various ergot alkaloids, such as LSD. The toxic mechanism of LSD is likely related to perturbations of serotonergic neurotransmission, resulting in profound alterations in perception, mood, and judgment as well as hallucinogenic effects in humans. This effect appears to be mediated by the strong partial agonist effect at the 5-HT2A (serotonin) receptor with an increase of glutamate release in the cerebral cortex. Catecholaminergic stimulation also occurs, resulting in mydriasis, increased blood pressure, tachycardia, elevated body temperature, tremors, and hyperreflexia. Ergot alkaloids are not teratogenic and are not considered genotoxic. There has only been one published carcinogenesis study performed in the rat using a crude ergot extract in the 1940s. A non-genotoxic mechanism was proposed for the aural neurofibromas found in this study. Species Susceptibility In laboratory animals ergot alkaloids exhibit moderate oral acute toxicity, with the rabbit much more sensitive than the rat and mouse to both per os and iv administration. While all clinical syndromes can be seen in most species, including humans, there are species differences for the most commonly encountered syndromes. The gangrenous form can affect all domestic animals, birds, and humans, but is most commonly seen in cattle. The hyperthermic syndrome is most commonly seen in cattle and the reproductive form (hypoprolactinemia) in pregnant mares pastured on endophyte-infected tall fescue. The horse and the pig are the most sensitive species to ergot alkaloid-induced hypoprolactinemia with resultant agalactia. Cattle are considered the next most sensitive, followed by sheep, with decreased milk production as a manifestation of ergot alkaloid toxicity. Total dietary ergovaline concentrations of > 100–200 ppb on a dry weight basis are considered potentially toxic for cattle and horses. This toxicity is dependent on other contributory factors such as breed, physiologic state, and temperature stress. Brahman-type cattle (Bos primigenus indicus) are more resistant than European breeds (Bos taurus) to both heat stress and other aspects of fescue toxicosis. A primary enteric form has been described in humans ingesting grain contaminated with

Claviceps fusiformis that produces the clavine alkaloids. The NOAEL for ergot alkaloids in the piglet is 0.15 mg/kg feed. Poultry appear fairly resistant to ergot alkaloid effects, with a NOAEL of 1.4 mg/kg feed. Individual responses vary with the total amount and mixture of individual alkaloids in the feed, frequency of ingestion, climatic (cool, wet weather is associated with apparent increases in the risk of gangrenous lesions), and other conditions.

7.3. Manifestations of Toxicity in Animals Clinically, ergot alkaloid toxicosis can manifest as gangrenous, hyperthermic, reproductive, neurologic, and enteric syndromes. The specific clinical syndrome manifested depends on the ergot alkaloid or combination present, amount consumed, period of exposure, species, age and reproductive status, genetics, ambient temperature, and nutrition. More than one syndrome may manifest at one time. Laboratory Animals Sublethal acute exposure to ergot alkaloids induces signs of neurotoxicity, including restlessness, miosis or mydriasis, muscular weakness, tremor, and rigidity. Repeated doses result in ischemic lesions, especially in the extremities, decreased body weight gain, and changes in some hormones, especially prolactin. Ischemic lesions leading to necrosis occur in the tail of rats, the comb and wattles of cockerels, and the margins of the external ear in dogs and rabbits. The NOAEL for the rat for repeated exposure to several different ergot alkaloids is approximately 0.22–0.6 mg/kg bw per day, with tail muscle atrophy, presumably due to vasoconstrictive effects, as the endpoint. Reproductive effects include interference with implantation, embryotoxicity, and inhibition of lactation. Livestock Fescue toxicoses in livestock grazing endophyte-infected tall fescue or hay can manifest as dry gangrene of the extremities in cattle, hyperthermia and production loss in cattle, agalactia and reproductive losses in horses, and, infrequently, neurological effects (see below). These syndromes are the effects of subacute or chronic exposure to ergopeptine alkaloids, primarily

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ergovaline at  200–600 ppm. Gangrene and reproductive effects can also occur following exposure to Claviceps purpurea-infected grains and grasses. Unlike in fescue toxicosis, gangrene is generally not temperature dependent, and reproductive effects tend to occur earlier in pregnancy and manifest as abortions due to the presence of ergonovine, also called ergometrine, because of its oxytocic effect. Hyperthermia has also been described in cattle with toxicoses due to contamination of grains and grasses by several different Claviceps spp., including C. africana, C. cyperi, C. purpurea, and C. sorghi. Dual infection of tall fescue by both Claviceps purpurea and Neoptyphodium coenophialum has been reported. GANGRENOUS SYNDROME

The gangrenous syndrome occurs most commonly in cattle, and is called fescue foot if cattle have been grazing on endophyte-infected tall fescue. Fescue foot often follows the onset of cooler or cold weather (below 8
C [46
F] and especially in the presence of snow or ice), when blood supply to the extremities tends to be reduced. Gangrene following exposure to Claviceps purpurea-infected grains and grasses is generally not temperature dependent. The gangrenous form can occur when animals ingest ergot alkaloids over a period of days (5–15) or weeks. Clinical signs include reduced feed intake, unthriftiness, lameness, swelling, and sloughing of feet below the fetlocks (generally the hind feet). Affected extremities may become inflamed and then cold, with numbness and dry gangrene developing. Usually a line of demarcation separates the proximal viable tissue and the distal dry, cold epidermis. If the legs are affected, the line of demarcation is often at the coronary band, which becomes swollen (Figure 39.19). Eventually, there is sloughing of distal tissue. Less commonly, the ears and tail are sloughed. Vascular lesions as described for humans can be found. Cutaneous ergotism with skin necrosis resembling photosensitization has been reported. Similar clinical signs have been reported from an outbreak in water buffalo (Bubalus bubalis) raised for meat in the UK, and in free-living moose from Norway. In sheep, gangrene of the tongue also may be seen. In poultry, comb gangrene is a major symptom following ergot

FIGURE 39.19 Gangrenous syndrome, field case of fescue foot, cow. There is marked reddening and swelling at the coronary band. Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M. Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 18, p. 688, with permission.

alkaloid toxicosis. Gangrene of toes and beak, as well as weight loss and general unthriftiness, may also occur. HYPERTHERMIC SYNDROME (SUMMER FESCUE TOXICOSIS, SUMMER SLUMP, SYSTEMIC HYPERTHERMIA)

This syndrome is seen primarily in cattle pastured on endophyte-infested tall fescue, especially when summer temperatures exceed 31
C (88
F). It is characterized by hyperthermia, poor weight gain, reduced feed intake, reduced conception rates, intolerance to heat, a failure to shed the winter hair coat (rough hair), nervousness, and excessive salivation. Decreased milk production can lead to decreased weaning weight of calves. The vasoconstrictive effects of ergot alkaloids interfere with the normal physiological response to heat stress, which consists of dissipation of heat from the body surface by way of increased

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blood flow to the periphery, where surface blood vessels are dilated. Instead, there is reduced blood flow to peripheral tissues and heat does not dissipate normally, leading to elevated body temperature. Mortality is negligible. Rabbits are very sensitive to hyperthermia induced by ergot alkaloids. REPRODUCTIVE SYNDROME

Horses and swine are the most sensitive species to ergot alkaloid-induced hypoprolactinemia. The primary signs in swine showing ergot alkaloid toxicosis are agalactia, early parturition, weak or dead piglets, infertility, and reduced rate of weight gain. In horses, the effects of ergopeptine alkaloids are manifested late in pregnancy, generally when mares consume endophyte-infected grass after day 300 of pregnancy and especially during the last 30 days. Pregnant mares are sensitive to ergopeptine alkaloid levels as low as 50–100 mg/kg in the diet. Serum cortisol concentrations, progestagens (mainly 5a pregnanes) and prolactin do not increase normally near parturition, and triiodothyronine levels are extremely low. Mares may abort or, more often, have dystocia after prolonged gestation. A tough, thickened, and edematous placenta may be present that increases the need for assistance during parturition. Chorioallantoic changes include edema, smooth muscle hyperplasia, mucoid degeneration of vessels, and fibrosis (Figure 39.20), and are consistent with anoxia due to vasoconstriction. The low progestagen levels may contribute to the prolonged gestation and to poor adrenocortical function in foals that are born small or weak, or are stillborn. Foals may be carried past term and be large and weak at birth with overgrown hooves and prematurely erupted teeth. Survival of foals is very low and mortality increases in mares, especially if they are not assisted during parturition. Foals may also be premature and small. Affected mares have low milk production or agalactia attributable to the low prolactin levels. Fetal membranes may be retained. Other clinical signs in pregnant mares grazing toxic pastures are intermittent diarrhea and excessive sweating. Reproduction and lactation problems also affect cattle and sheep pastured on endophyteinfected tall fescue. General reproductive efficiency is impaired, but without the pronounced

FIGURE 39.20 Placenta from a mare pastured on endophyte infected fescue grass with prolonged gestation. H&E stain. (A) There is thickening of the chorioallantois due to marked expansion of the vascular and connective tissue compartment of the allantois (AVC) and thickening of the allantoic membrane (AM). The villi of the chorion (C) are shortened. (B) Higher magnification of (A). Allantoic vessels have undergone smooth muscle hypertrophy with mucinous degeneration, and are separated by mucinous material and edema.

effect on the fetus and parturition that occur in horses. This is because cattle have a placental lactogen during parturition so they are not as dependent on prolactin for lactation as horses. In cattle, reduced calving rates (70–80% calf crop) may be due to altered luteal function and decreased circulating progesterone occurring in early pregnancy. Dairy cows may have a marked decrease in milk production due, in part, to depressed prolactin concentrations. In ewes, serum prolactin levels are depressed by ingestion of affected fescue and there is an increased rate of return to estrus.

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FAT NECROSIS

Fat necrosis or lipomatosis can occur in cattle pastured on endophyte-infected tall fescue over several seasons. It is characterized by the presence of multiple hard, yellowish or chalky white irregularly shaped masses in the abdominal adipose tissue, most notably in the mesenteries; subcutaneous fat is not affected. On cut section, the masses have a dry, hard, cheesy, opaque appearance; calcification occasionally may be present. Causative factors may be vasoconstriction that occurs either directly or secondarily to a febrile condition, or increased oxidative stress that can trigger the lipolytic process. Because of its sporadic nature, this condition is of limited economic importance when sizable herds are encountered. Consequences of fat necrosis include digestive disturbances due to obstruction or intestinal constriction, scanty feces, bloating, and, occasionally, death. Dystocia, due to hardening of adipose tissue around the birth canal, or urine retention, due to similar deposits along the urinary tract, with associated postrenal uremia, may occur. Other signs associated with fat necrosis include weight loss, poor appetite, listlessness, and rough hair coat. The masses associated with fat necrosis may be detected on rectal palpation. NEUROLOGIC (CONVULSIVE) SYNDROME

This syndrome occurs in cattle and horses due to Claviceps paspali infection of Paspalum spp. grasses (e.g., dallisgrass). Clinical signs, including nervousness and stamping of the feet, develop in animals kept on contaminated hay after about 1 week. They progress to include hyperexcitability, belligerency, incoordination, convulsions, and opisthotonus (star-gazing posture).

7.4. Human Risk and Disease Epidemics of human ergotism occurred in Europe in the Middle Ages, and specifically in France from the 9th to the 14th centuries due to the consumption of ergot (Claviceps spp. infected) in the grain. Manifestations of ergotism included itching, numbness, muscle cramps, sustained spasms and convulsions, and extreme pain. A victim’s extremities, usually a foot or leg, would feel cold, alternating with a burning sensation (St Anthony’s fire). Numbness and

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dry gangrene, followed by loss of fingers, hands, or feet, was common. Whole limbs could become gangrenous and slough off. Abortion or agalactia in nursing mothers was a frequent complication of ergot poisoning. A convulsive form of ergotism was also known and characterized by convulsive episodes every few days as well as manic episodes and hallucinations. It has been suggested by the historian Mary Matossian that the frenzied activities of peasants that culminated in the French Revolution may have been due to the ergot alkaloids in rye bread, the staple food of the peasants at that time. It has been suggested that ergot alkaloids were also a factor contributing to the Salem witch trials in the United States. With changes in farming practices, such as wheat replacing rye as the major grain crop, the epidemics ceased. Current human risk from ergot alkaloids is primarily from ingestion of contaminated grains or by-products, or from overdose of either therapeutic drugs or drugs of abuse, such as LSD. Due to implementation of regulations and advances in agricultural and milling procedures in developed countries, human risk in these countries is primarily from overdose of therapeutic drugs (e.g., ergotamine tartrate used for migraine headaches), from drug abuse (e.g., ingestion of large amounts of ergotamine to induce abortion), or from use of recreational drugs (e.g., LSD), rather than from ingestion of contaminated cereal grains. Several epidemics of ergotism in developing countries were reported. In India (1958–1975) following infection of pear millet (bajra) by Claviceps fusiformis. Symptoms were enteric, including nausea and emesis; giddiness was also reported. Two epidemics of gangrenous ergotism were reported (1978 and 2001) in Ethiopia due to Claviceps purpurea sclerotia from wild oats contaminating barley (0.75% ergot). Ergotamine and ergometrine were detected. General symptoms included weakness, formication (a tactile hallucination involving the belief that something is crawling on the body or under the skin), burning sensation, nausea, emesis, and diarrhea. Infants died from starvation, presumably due to lactation problems. Although epidemics of ergotism do not occur in developed countries, contamination of grain by Claviceps spp. is still common. In the USA, wheat and rye are considered unsafe for human

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consumption if they contain more than 0.3% sclerotia by weight, and oats, triticale, or barley are so graded when they contain more than 0.1%. The maximum level for ergot set by the EU is 0.05% in durum and common wheat, i.e., 0.05% or 500 mg/kg w/w sclerotia. A similar level is set for cereal grains in Australia and New Zealand. In Canada, cereal grains are graded ranging from 0.01% ergot sclerotia for the highest quality to 0.1% for the lowest quality. Within the EU, the ergot group TDI is 0.06 mg/ kg bw per day and the group acute reference dose (ARfD) is 1 mg/kg bw for ergot alkaloids. The highest dietary exposure occurs in those countries with relatively high consumption of rye bread and rolls, especially in toddlers and children. Maximum levels for ergot alkaloids in animal feed are 3 mg/kg for cattle, sheep, and horses, 6 mg/kg for pigs, and 3 mg/kg for poultry. Side effects of therapeutic usage of ergotamine in migraine preparations include nausea and, less commonly, abdominal and muscle cramps of the lower extremities, diarrhea, and vertigo. At high doses, acute effects include emesis, diarrhea, intense thirst, itching, tingling and cold skin, rapid and weak pulse, confusion, and coma. Death may follow. The most common serious chronic effect reported is ischemia of the extremities due to severe peripheral vasospasm (demonstrated by angiography), resulting in gangrene. The acute effects of ergotamine overdose in humans are due to its marked vasoconstrictor effects. Emesis occurs due to stimulation of central dopamine receptors. Chronic administration results in cardiovascular lesions (see Cardiac, Vascular, and Skeletal Muscle Systems, Chapter 46). Medial (smooth muscle) hypertrophy and hyperplasia with intimal proliferation, characterized by influx and proliferation of smooth muscle cells, increased glycosaminoglycan production, and endothelial cell hypertrophy, occurs in arteries and arterioles, leading to occlusive vascular lesions in peripheral vessels. Proliferative valvulopathy may occur due to stimulation of stromal cells in the cardiac valves via the 5HT2B serotonin receptor. Peripheral neuropathy with atrophy of affected motor units has also been described. Overuse of ergotamine has also been reported to result in encephalopathy, focal motor or sensory symptoms, seizures, and coma.

The powerful oxytocic action of ergotamine on the pregnant uterus results in smooth muscle contractions. Use of ergotamine for its abortifacient effects frequently led to excessive uterine contraction and often caused ischemic damage to the fetus; hence, ergotamine is now contraindicated during pregnancy. However, ergometrine maleate is prescribed in the active management of third-stage labor and in prevention or treatment of postpartum hemorrhage. Overdose of ergometrine can result in seizures and gangrene. LSD use causes signs of catecholaminergic stimulation, such as mydriasis, increased blood pressure, tachycardia, elevated body temperature, tremor, and hyperreflexia, as well as severe perceptual distortion and hallucinations. The effects generally disappear over a 12-hour period. Long-term use may precipitate persistent psychosis or post-hallucinogenic perceptual disorder.

7.5. Diagnosis, Treatment and Prevention Presumptive diagnosis of ergotism is usually based on clinical signs and evidence of exposure. Screening for ergot sclerotia in cereal grains can be achieved by visual inspection, by near infrared hyperspectral imaging, or by determination of ricinoleic acid by GC-flame ionization detector (FID). Sclerotia in grasses are generally readily detectable by visual examination. By shaking several handfuls over a plastic bag and examining the chaff, it is possible to find sclerotia in baled forages. However microscopic examination of ground diets (feed microscopy) may be needed to determine the presence of ergot. Individual ergot alkaloids in food and feed commodities may be identified by high performance liquid chromatography with fluorescence detection (HPLC-FLD) and tandem-mass spectrometry (HPLC-MS/MS); the HPLC-FLD method has been internationally validated for the 12 most important ergot alkaloids found in grain and flour. In cases of ergot alkaloid toxicity associated with endophyte-infected tall fescue, fescue tillers (large grass shafts cut at the level of the soil) can be submitted for determination of the percent infection by the endophyte. Grass or hay should be analyzed for ergovaline and other ergopeptine alkaloids by HPLC-MS, or for total ergot alkaloids by competitive ELISA. Urinary alkaloid

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excretion and serum prolactin levels are useful diagnostic tests. In humans, vasoconstrictive effects have been treated with anticoagulants, low molecular weight dextran, and nitroprusside. In livestock, treatment consists of isolating animals from the source and, if appropriate, moving them to a dry, warm environment to avoid aggravation of the vascular insult. Intravenous infusion of sodium nitroprusside, a potent vasodilator, has also been used to alleviate the vasoconstriction. In horses, treatment of agalactia and prolonged gestation is possible using D2 dopamine receptor antagonists, such as domperidone or sulpiride, either before or after parturition. The risk of ergot alkaloid toxicity from grains is decreased by mechanical seed cleaning and sorting, which decreases the presence of sclerotia. Milling processes redistribute sclerotia particles in different milling fractions. Processing grain, in particular baking, decreases the total amount of ergot alkaloids to some extent, and changes the ratio between epimeric forms. In the case of endophyte-infected tall fescue pastures or hay, prevention can be largely accomplished by husbandry measures such as avoiding or decreasing exposure of animals, especially where seeds are present. Pregnant mares should be removed from infected pasture by day 300 of gestation. Ammoniation of hay will reduce toxicity, and detoxification is dependent on ammonia concentrations. Ensiling and storage can reduce toxicity.

8. SUMMARY/CONCLUSION Mycotoxins are a structurally and functionally diverse group of organic compounds that affect humans and animals. They can affect all organ systems, but individual mycotoxins usually target specific organ systems. Species differences are generally related to severity of effect, although in some cases target organs may differ according to species. Some mycotoxins cause primarily acute and highly reversible effects, others cause irreversible organ damage, and still others cause both acute and chronic effects, depending upon exposure levels, time course, and other circumstances. Recognizing the characteristics of the clinical and morphologic manifestations, the time course of onset, potential

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reversibility, and linkage to sufficiently contaminated source materials (most often a foodstuff) is essential in the diagnosis of mycotoxicoses. Biomarkers for exposure to selected mycotoxins are available. Although mycotoxin-induced diseases have been recognized for many years, even centuries, recognition of specific mycotoxins as causes of disease is a comparatively recent development. As a result, major knowledge gaps remain to be filled through research for many of the known mycotoxins, particularly the potential human health effects of low-level chronic exposure, identification of biomarkers of exposure, and the effects of mycotoxin interactions. Mycotoxins continue to attract worldwide attention because of their impacts on human and animal health, agricultural losses, and the potential effects of climate change.

SUGGESTED READING General Berthiller, F., Crews, C., Dall’asta, C., Saeger, S.D., Haesaert, G., Karlovsky, P., Oswald, I.P., Seefelder, W., Speijers, G., Stroka, J., 2013. Masked mycotoxins: A Review. Mol. Nutr. Food Res. 57, 165–186. Bhat, R.V., Shetty, P.H., Amruth, R.P., Sudershan, R.V., 1997. A foodborne disease outbreak due to the consumption of moldy sorghum and maize containing fumonisin mycotoxins. J. Toxicol. Clin. Toxicol. 35, 249–255. Bondy, G.S., Pestka, J.J., 2000. Immunomodulation by fungal toxins. J. Toxicol. Environ. Health B, Crit. Rev. 3, 109–143. Bryden, W.L., 2012. Mycotoxin contamination of the feed supply chain: implications for animal productivity and feed security. Anim. Feed Sci. Technol. 173, 134–158. CAST Task Force Report 139, 2003. Mycotoxins: Risks in Plant, Animal, and Human Systems. Council for Agricultural Science and Technology, Ames, IA. 2003. Eaton, D.L., Beima, K.M., Bammler, T.K., Riley, R.T., Voss, K.A., 2010. Hepatotoxic mycotoxins. In: Roth, R.A., Ganey, P.E. (Eds.) Comprehensive Toxicology, second ed. vol. 10. Elsevier, New York, NY, pp. 527–569. IARC-WHO, 1993. Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC. Monographs on the Evaluation of Carcinogenic Risks to Humans 56, 467–488. Jestoi, M., 2008. Emerging fusarium-mycotoxins, fusaproliferin, beauvericin, enniatins, and moniliformin: a review. Crit. Rev. Food. Sci. Nutr. 48, 21–49. Ko¨ppen, R., Koch, M., Siegel, D., Merkel, S., Maul, R., Nehls, I., 2010. Determination of mycotoxins in foods: current state of analytical methods and limitations. Appl. Microbiol. Biotechnol. 86, 1595–1612.

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Kuiper-Goodman, T., 1998. Food safety: mycotoxins and phycotoxins in perspective. In: Miraglia, M., van Egmond, H., Brera, C., Gilbert, J. (Eds.), Mycotoxins and Phycotoxins – Developments in Chemistry, Toxicology, and Food Safety. Fort Collins, Alaken Inc., CO, pp. 25–48. Leung, M.C., Dı´az-Llano, G., Smith, T.K., 2006. Mycotoxins in pet food: a review on worldwide prevalence and preventative strategies. J. Agric. Food. Chem. 54, 9623–9635. Matossian, M.A.K., 1989. Poisons of the Past: Molds: Epidemics and History. Yale University Press, New Haven, CT. Meggs, W.J., 2009. Epidemics of mold poisoning: past and present. Toxicol. Ind. Health 25, 571–576. Richard, J.L., 2007. Some major mycotoxins and their mycotoxicoses – an overview. Int. J. Food Microbiol. 119, 3–10. Riley, R.T., Voss, K.A., 2011. Developing mechanism-based and exposure biomarkers for mycotoxins in animals. In: De Saeger, S. (Ed.), Determining Mycotoxins and Mycotoxigenic Fungi in Food and Feed. Ghent University, Woodhead Publishing Limited, Cambridge, UK, pp. 245–278. Scudamore, K.A., Hetmanski, M.T., Nawaz, S., Naylor, J., Rainbird, S., 1997. Determination of mycotoxins in pet foods sold for domestic pets and wild birds using linkedcolumn immunoassay clean-up and HPLC. Food Addit. Contam. 14, 175–186. Snyman, L.D., Kellerman, T.S., Vleggaar, R., Flett, B.C., Basson, K.M., Schultz, R.A., 2011. Diplonine, a neurotoxin isolated from cultures of the fungus Stenocarpella maydis (Berk.) Sacc. that induces diplodiosis. J. Agric. Food Chem. 59, 9039–9044. Turner, P.C., Flannery, B., Isitt, C., Ali, M., Pestka, J., 2012. The role of biomarkers in evaluating human health concerns from fungal contaminants in food. Nutr. Res. Rev. 25, 162–179. World Health Organization (WHO), 1990. Selected Mycotoxins: Ochratoxins, Trichothecenes, Ergot. Environmental Health Criteria; 105, United Nations Environmental Programme. International Labour Organization and the World Health Organization, Geneva. Wild, C.P., Gong, Y.Y., 2010. Mycotoxins and human disease: a largely ignored global health issue. Carcinogenesis 31, 71–82.

Aflatoxins Azziz-Baumgartner, E., Lindblade, K., Gieseker, K., Rogers, H.S., Kieszak, S., Njapau, H., Schleicher, R., McCoy, L.F., Misore, A., DeCock, K., Rubin, C., Slutsker, L., 2005. Aflatoxin Investigative Group. Case–control study of an acute aflatoxicosis outbreak, Kenya, 2004. Environ. Health. Perspect. 113, 1779–1783. Erratum in: Environ. Health Perspect. (2006). 114, A90. Caloni, F., Cortinovis, C., 2011. Toxicological effects of aflatoxins in horses. Vet. J. 188, 270–273. Coppock, R.W., Christian, R.R.G., Jacobsen, B.J., 2012. Aflatoxins. In: Gupta, R.C. (Ed.), Veterinary Toxicology, second ed. Elsevier, New York, NY, pp. 1181–1191.

Dereszynski, D.M., Center, S.A., Randolph, J.F., Brooks, M.B., Hadden, A.G., Palyada, K.S., McDonough, S.P., Messick, J., Stokol, T., Bischoff, K.L., Gluckman, S., Sanders, S.Y., 2008. Clinical and clinicopathological features of dogs that consumed foodborne hepatotoxic aflatoxins: 72 cases (2005–2006). J. Am. Vet. Med. Assoc. 232, 1329–1337. El-Serag, H.B., 2012. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 142, 1264–1273. Gouas, D., Shi, H., Hainaut, P., 2009. The aflatoxin-induced TP53 mutation at codon 249 (R249S): biomarker of exposure, early detection and target for therapy. Cancer Lett. 286, 29–37. Gross-Stainmeyer, K., Eaton, D.L., 2012. Dietary modulation of the biotransformation and genotoxicity of aflatoxin B1. Toxicology 299, 69–79. Hoerr, F.J., D’Andrea, G.H., Giambrone, J.J., Panangala, V.S., 1986. Comparative histopathologic changes in aflatoxicosis. In: Richard, J.L., Thurston, J.R. (Eds.), Diagnosis of Mycotoxicoses. Martin Nijhoff Publishers, Boston, MA, pp. 179–189. Jiang, Y., Jolly, P.E., Ellis, W.O., Wang, J.S., Phillips, T.D., Williams, J.H., 2005. Aflatoxin B1 albumin adduct levels and cellular immune status in Ghanaians. Int. Immunol. 17, 807–814. Jolly, P.E., Shuaib, F.M., Jiang, Y., Preko, P., Baidoo, J., Stiles, J.K., Wang, Phillips, J.S., Williams, J.H., 2011. Association of high viral load and abnormal liver function with high aflatoxin B1-albumin adduct levels in HIV-positive Ghanaians. Food Addit. Contam. Part A Chem. Anal. Control. Expo. Risk. Assess. 28, 1224–1234. Kensler, T.W., Egner, P.A., Wang, J.B., Zhu, Y.R., Zhang, B.C., Lu, P.X., Chen, J.G., Qian, G.S., Kuang, S.Y., Jackson, P.S., Gange, S.J., Jacobson, L.P., Mun˜oz, A., Groopman, J.D., 2004. Chemopreventation of hepatocellular carcinoma in aflatoxin endemic areas. Gestroenterology 127, S310–S318. Kensler, T.W., Roebuck, B.D., Wogan, G.N., Groopman, J.D., 2011. Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. Toxicol. Sci. 120 (S1), S28–S48. Khlangwiset, P., Shephard, G.S., Wu, F., 2011. Aflatoxins and growth impairment: a review. Crit. Rev. Toxicol. 41, 740–755. Liu, Y., Wu, F., 2010. Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ. Health Perspect. 118, 818–824. Liu, Y., Chang, C.C., March, G.M., Wu, F., 2012. Population attributable risk of aflatoxin-related liver cancer: systematic review and meta-analysis. Eur. J. Cancer 48, 2125–2136. Liu, Y., Wu, F., 2010. Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ. Health Perspect. 118, 818–824. Miller, D.M., Crowell, W.A., Stuart, B.P., 1982. Acute aflatoxicosis in swine: clinical pathology, histopathology, and electron microscopy. Am. J. Vet. Res. 43, 273–277. Newberne, P.M., Butler, W.H., 1969. Acute and chronic effects of aflatoxin on the liver of domestic and laboratory animals: a review. Cancer Res. 29, 236–250.

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Newberne, P.M., Wogan, G.N., 1968. Sequential morphological changes in aflatoxin B1 carcinogenesis in the rat. Cancer Res. 28, 770–781. Newman, S.J., Smith, J.R., Stanske, K.A., Newman, L.B., Dunlap, J.R., Imerman, P.M., Kirk, C.A., 2007. Aflatoxicosis in nine dogs after exposure to contaminated commercial dog food. J. Vet. Diag. Invest. 19, 168–175. Rawal, S., Kim, J.E., Coulombe Jr., R., 2010. Aflatoxin B1 in poultry: toxicology, metabolism and prevention. Res. Vet. Sci. 89, 325–331. Shuaib, F.M., Jolly, P.E., Ehiri, J.E., Yatich, N., Jiang, Y., Funkhouser, E., Person, S.D., Wilson, C., Ellis, W.O., Wang, J.S., Williams, J.H., 2010. Association between birth outcomes and aflatoxin B1 biomarker blood levels in pregnant women in Kumasi, Ghana. Trop. Med. Int. Health 15, 160–167. Stenske, K.A., Smith, J.R., Newman, S.J., Newman, L.B., Kirk, C.A., 2006. Aflatoxicosis in dogs and dealing with suspected contaminated commercial foods. J. Am. Vet. Med. Assoc. 228, 1686–1691. Sun, Z., Lu, P., Gail, M.H., Pee, D., Zhang, Q., Ming, L., Wang, J., Wu, Y., Lui, G., Wu, Y., Zhu, Y., 1999. Increased risk of hepatocellular carcinoma in male hepatitis B surface antigen carriers with chronic hepatitis who have detectable aflatoxin metabolite M1. Hepatology 30, 379–383. Turner, P.C., Moore, S.E., Hall, A.J., Prentice, A.M., Wild, P.C., 2003. Modification of immune function through exposure to dietary aflatoxin in Gambian children. Environ. Health Perspect. 111, 217–220. Turner, P.C., Collinson, A.C., Cheung, Y.B., Gong, Y., Hall, A.J., Prentice, A.M., Wild, P.C., 2007. Aflatoxin exposure in utero causes growth faltering in Gambian infants. Int. J. Epidemiol. 36, 1119–1125. Villar, S., Ortiz-Cuaran, S., Abedi-Ardekani, B., Gouas, D., Nogueira da Costa, A., Plymoth, A., Khuhaprema, T., Kalalak, A., Sangrajrang, S., Friesen, M.D., Groopman, J.D., Hainaut, P., 2012. Aflatoxin-induced TP53 R249S mutation in hepatocellular carcinoma in Thailand: association with tumors developing in the absence of liver cirrhosis. PLoS One 7, e37707. Wild, C.P., Montesano, R., 2009. A model of interaction: aflatoxin and hepatitis viruses in cancer aetiology and prevention. Cancer Lett. 286, 22–28. Williams, D.E., 2012. The rainbow trout liver cancer model: response to environmental chemicals and studies on promotion and chemoprevention. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155, 121–127. Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., Aggarwal, A., 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am. J. Clin. Nutr. 80 1106–1022. Wogan, G.N., Kensler, T.W., Goopman, J.D., 2012. Present and future directions of translational research on aflatoxin and hepatocellular carcinoma: a review. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29, 249–257.

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a biomarker of environmental exposure to aristolochic acid. Kidney Int. 81, 559–567. Jung, K.Y., Takeda, M., Kim, D.K., Tojo, A., Narikawa, S., Yoo, B.S., Hosoyamada, M., Cha, S.H., Sekine, T., Endou, H., 2001. Characterization of ochratoxin A transport by human organic anion transporters. Life Sci. 21, 2123–2135. Kozaczynski, W., 1994. Experimental ochratoxicosis A in chickens. Histopathological and histochemical study. Arch. Vet. Pol. 34, 205–219. Kuiper-Goodman, T., Hilts, C., Billiard, S.M., Kiparissis, Y., Richard, I.D.K., Hayward, S., 2010. Health risk assessment of ochratoxin A for all age-sex strata in a market economy. Food Addit. Contam. Part A. Chem. Anal. Control Expo. Risk Assess. 27, 212–240. Mally, A., 2012. Ochratoxin A and mitotic disruption: mode of action analysis of renal tumor formation by ochratoxin A. Toxicol. Sci. 127, 315–330. Mally, A., Dekant, W., 2009. Mycotoxins and the kidney: modes of action for renal tumor formation by ochratoxin A in rodents. Mol. Nutr. Food Res. 53, 467–478. Mantle, P.G., Kulinskaya, E., 2010. Lifetime, low-dose ochratoxin A dietary study on renal carcinogenesis in male Fischer rats. Food Addit. Contam. Part A. Chem. Anal. Control Expo. Risk Assess. 27, 1566–1573. Mantle, P.G., Faucet-Marquis, V., Manderville, R.A., Squillaci, B., Pfohl-Leszkowicz, A., 2010. Structures of covalent adducts between DNA and ochratoxin A: a new factor in the debate about genotoxicity and human risk assessment. Chem. Res. Toxicol. 23, 89–98. National Toxicology Program, 1989. Technical Report on the Toxicology and Carcinogenesis Studies of Ochratoxin A (Case No 303-47-9) in F344 N Rats. NTP Technical Report No. 358. In: Boorman, G. (Ed.), US Department of Health and Human Services. National Institutes of Health, Research Triangle Park, NC.  2010. “Suspects” in etiology of Pepeljnjak, S., Klaric, M. S, endemic nephropathy: aristolochic acid versus mycotoxins. Toxins (Basel) 2, 1414–1427.  c, M., 2008. Mycotoxin and Peraica, M., Domijan, A.-M., Sari aristolochic acid theories of the development of endemic nephropathy. Arh. Hig. Rada Toksikol. 59, 59–65. Pfohl-Leszkowicz, A., Manderville, R.A., 2011. An update on direct genotoxicity as a molecular mechanism of ochratoxin A carcinogenicity. Chem. Res. Toxicol. 25, 252–262. Rached, E., Hard, G.C., Blumbach, K., Weber, K., Draheim, R., Lutz, W.K., Ozden, S., Steger, U., Dekant, W., Mally, A., 2007. Ochratoxin A: 13-week oral toxicity and cell proliferation in male F344 rats. Toxicol. Sci. 97, 288–298. Stoev, S.D., Hald, B., Mantle, P.G., 1998. Porcine nephropathy in Bulgaria: a progressive syndrome of complex or uncertain (mycotoxin) aetiology. Vet. Rec. 142, 190–194. Szczech, G.M., Carlton, W.W., Tuite, J., 1973a. Ochratoxicosis in beagle dogs. II Pathology. Vet. Pathol. 10, 219–231. Szczech, G.M., Carlton, W.W., Tuite, J., Caldwell, R., 1973b. Ochratoxin A toxicosis in swine. Vet. Pathol. 16, 466–475.

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Wei, X., Sulik, K.K., 1993. Pathogenesis of craniofacial and body wall malformations induced by ochratoxin A in mice. Am. J. Med. Genet. 47, 862–871. Wu, Q., Dohnal, V., Huang, L., Kuca, K., Wang, X., Chen, G., Yuan, Z., 2011. Metabolic pathways of ochratoxin A. Curr. Drug. Metab. 12, 1–10. Zlender, V., Breljak, D., Ljubojevic, M., Flajs, D., Baleln, D., Brzica, H., Domijan, A.M., Peraica, M., Fuchs, R., Anzai, N., Sabolic, I., 2009. 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. 239, 284–296.

Trichothecenes General Beasley, V.R. (Ed.), 1989. Trichothecene Mycotoxicosis: Pathophysiologic Effects. CRC Press, Boca Raton, FL. Cresia, D.A., Lambert, R.J., 1989. Acute respiratory tract toxicity of the trichothecene mycotoxin, T-2 toxin. In: Beasley, V.R. (Ed.), Trichothecene Mycotoxicosis: Pathophysiological Effects, vol. 1. CRC Press, Inc., Boca Raton, FL, pp. 161–170. Haschek, W.M., Beasley, V.R., 2009. Trichothecene mycotoxins. In: Gupta, R.C. (Ed.), Handbook of Toxicology of Chemical Warfare Agents. Academic Press, Elsevier, San Diego, CA, pp. 353–369. McCormick, S.P., Stanley, A.M., Stover, N.A., Alexander, N.J., 2011. Trichothecenes: from simple to complex mycotoxins. Toxins (Basel) 3 (7), 802–814. Mostrom, M.S., Raisbeck, M.F., 2012. Trichothecenes. In: Gupta, R.C. (Ed.), Veterinary Toxicology, second ed. Academic Press, San Diego, CA, pp. 1239–1265. Schollenberger, M., Drochner, W., Mu¨ller, H.M., 2007. Fusarium toxins of the scirpentriol subgroup: a review. Mycopathologia 164 (3), 101–118. Sudakin, D.L., 2003. Trichothecenes in the environment: relevance to human health. Toxicol. Lett. 143, 97–107. Trenholm, H.L., Friend, D., Hamilton, R.M.G., Prelusky, D.B., Foster, B.C., 1989. Lethal toxicity and nonspecific effects. In: Beasley, V.R. (Ed.), Trichothecene Mycotoxicosis: Pathophysiological Effects, vol. 1. CRC Press, Inc., Boca Raton, FL, pp. 161–170. Ueno, Y., 1985. The toxicology of mycotoxins. Crit. Rev. Toxicol. 14 (2), 99–132. Wu, Q., Dohnal, V., Huang, L., Kuca, K., Yuan, Z., 2010. Metabolic pathways of trichothecenes. Drug. Metab. Rev. 42, 250–267.

Deoxynivalenol Charmley, E., Trenholm, H.L., Thompson, B.K., Vudathala, D., Nicholson, J.W.G., Prelusky, D.B., Charmley, L.L., 1993. Influence of level of deoxynivalenol in the diet of dairy cows on feed intake, milk production, and its composition. J. Dairy Sci. 76, 3580–3587.

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T-2 Toxin European Food Safety Authority Panel on Contaminants in the Food Chain (CONTAM), 2011. Scientific opinion on the risks for animal and public health related to the presence of T-2 and HT-2 toxin in food and feed. EFSA J. 9, 2418 [187 pages]. Available at www.efsa.europa.eu/efsajournal (last accessed 10/2012). Ihara, T., Sugamata, M., Sekijima, M., Okumura, H., Yoshino, N., Ueno, Y., 1997. Apoptotic cellular damage in mice after T-2 toxin-induced acute toxicosis. Nat. Toxins 5, 141–145. Joffe, A.Z., 1983. Food borne diseases: Alimentary toxic aleukia. In: Recheigl, M. (Ed.), CRC Handbook of Food-borne Diseases of Biological Origin. CRC Press, Boca Raton, FL, pp. 353–495. Li, Y., Wang, Z., Beier, R.C., Shen, J., De Smet, D., De Saeger, S., Zhang, S., 2011. T-2 toxin, a trichothecene mycotoxin: review of toxicity, metabolism, and analytical methods. J. Agric. Food Chem. 59, 3441–3453. Pang, V.F., Adams, J.H., Beasley, V.R., Buck, W.B., Haschek, W.M., 1986. Myocardial and pancreatic lesions induced by T-2 toxin, a trichothecene mycotoxin, in swine. Vet. Pathol. 23, 310–319. Ravindran, J., Agrawal, M., Gupta, N., Rao, P.V., 2011. Alteration of blood brain barrier permeability by T-2 toxin: Role of MMP-9 and inflammatory cytokines. Toxicology 280, 44–52. Wang, L.H., Fu, Y., Shi, Y.X., Wang, W.G., 2011. T-2 toxin induces degenerative articular changes in rodents: link to Kaschin-Beck disease. Toxicol. Pathol. 39, 502–507. Yang, J.Y., Zhang, Y.F., Liang, A.M., Kong, X.F., Li, Y.X., Ma, K.W., Jing, A.H., Feng, S.Y., Qiao, X.L., 2010. Toxic effects of T-2 toxin on reproductive system in male mice. Toxicol. Ind. Health 26, 25–31.

Macrocyclic Trichothecenes Andersson, M.A., Nikulin, M., Koljalg, U., Andersson, M.C., Rainey, F., Reijula, K., Hintikka, E.L., Salkinoja-Salonen, M., 1997. Bacteria, molds, and toxins in water-damaged building materials. Appl. Environ. Microbiol. 63, 387–393. Carey, S.A., Plopper, C.G., Hyde, D.M., Islam, Z., Pestka, J.J., Harkema, J.R., 2012. Satratoxin-G from the black mold Stachybotrys chartarum induces rhinitis and apoptosis of olfactory sensory neurons in the nasal airways of rhesus monkeys. Toxicol. Pathol. 40, 887–898. Corps, K.N., Islam, Z., Pestka, J.J., Harkema, J.R., 2010. Neurotoxic, inflammatory, and mucosecretory responses in the nasal airways of mice repeatedly exposed to the

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Zearalenone Bandera, E.V., Chandran, U., Buckley, B., Lin, Y., Isukapalli, S., Marshall, I., King, M., Zarbl, H., 2011. Urinary mycoestrogens, body size and breast development in New Jersey girls. Sci. Total. Environ. 409, 5221–5227. Belli, P., Bellaton, C., Durand, J., Balleydier, S., Milhau, N., Mure, M., Mornex, J.F., Benahmed, M., Le Jan, C., 2010. Fetal and neonatal exposure to the mycotoxin zearalenone induces phenotypic alterations in adult rat mammary gland. Food Chem. Toxicol. 48, 2818–2826. Biehl, M.L., Prelusky, D.B., Koritz, G.D., Hartin, K.E., Buck, W.B., Trenholm, H.L., 1993. Biliary excretion and

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enterohepatic cycling of zearalenone in immature pigs. Toxicol. Appl. Pharmacol. 121, 152–159. Collins, T.F., Sprando, R.L., Black, T.N., Olejnik, N., Eppley, R.M., Alam, H.Z., Rorie, J., Ruggles, D.I., 2006. Effects of zearalenone on in utero development in rats. Food Chem. Toxicol. 44, 1455–1465. Dai, S., Duan, J., Lu, Y., Zhang, Y., Cheng, J., Ren, J., Zhao, X., Wu, Y., Yu, Y., Zuo, P., Wu, Y., Ge, Q., 2004. Phytoestrogen alph-ZEA ralanol inhibits atherogenesis and improves lipid profile in ovariectomized cholesterol-fed rabbits. Endocrine 25, 121–129. Da¨nicke, S., Swiech, E., Buraczewska, L., Ueberscha¨r, K.H., 2005. Kinetics and metabolism of zearalenone in young female pigs. J. Anim. Physiol. Anim. Nutr. (Berl.) 89, 268–276. European Food Safety Authority Panel on Contaminants in the Food Chain (CONTAM), 2011. Scientific Opinion on the risks for public health related to the presence of zearalenone in food. EFSA J. 9, 2197 [124 pp.]. Available at http://www.efsa.europa.eu/de/efsajournal/doc/2197.pdf (last accessed 10/2012). European Food Safety Authority, 2004. Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the Commission related to zearalenone as undesirable substance in animal feed. EFSA J. 89, 1–35. Fugh-Berman, A., 2003. “Bust enhancing” herbal products. Obstet. Gynecol. 101, 1345–1349. Gajecka, M., Janowski, T., Jakimiuk, E., Polak, M., PodhaliczDziegielewska, M., Rotkiewicz, T., Otrocka-Domaga1a, I., Obremski, K., Zielonka, L., Gajecki, M., 2007. Histopathological and immunohistochemical examinations, and changes in proliferation activity of the uterus in bitches following zearalenon micotoxicosis. Pol. J. Vet. Sci. 10, 143–151. Gajecka, M., Obremski, K., Jakimiuk, E., SkorskaWyszy nska, E., Zielonka, L., Gajecki, M., 2008. Histopathological examination of ovaries in bitches after experimental zearalenone mycotoxicosis. Pol. J. Vet. Sci. 11, 363–366. Gajecka, M., Rybarczyk, L., Zwierzchowski, W., Jakimiuk, E., Zielonka, L., Obremski, K., Gajecki, M., 2011. The effect of experimental, long-term exposure to low-dose zearalenone mycotoxicosis on the histological condition of ovaries in sexually immature gilts. Theriogenology 75, 1085–1094. Gajecka, M., Rybarczyk, L., Jakimiuk, E., Zielonka, q, Obremski, K., Zwierzchowski, W., Gajecki, M., 2012. The effect of experimental long-term exposure to low-dose zearalenone on uterine histology in sexually immature gilts. Exp. Toxicol. Pathol. 64, 537–542. Haschek, W.M., Haliburton, J.C., 1986. Diagnosis of Mycotoxicoses. In: Richard, J.L., Thurston, J.R. (Eds.), Fusarium moniliforme and zearalenone toxicoses in domestic animals: a review. Martin Nijhoff Publishers, Boston, MA, pp. 213–235. Jefferson, W.N., Padilla-Banks, E., Clark, G., Newbold, R.R., 2002. Assessing estrogenic activity of phytochemicals using transcriptional activation and immature mouse uterotrophic

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Ergot Alkaloids General Belser-Ehrlich, S., Harper, A.R., Hussey, J., Hallock, R.M., 2012. Human and cattle ergotism since 1900: Symptoms, outbreaks, and regulations. Toxicol. Ind. Health [Epub ahead of print]. Elenkova, A., Shabani, R., Kalinov, K., Zacharieva, S., 2012. Increased prevalence of subclinical cardiac valve fibrosis in patients with prolactinomas on long-term bromocriptine and cabergoline treatment. Eur. J. Endocrinol. 167, 17–25. European Food Safety Authority Panel on Contaminants in the Food Chain (CONTAM). (2012). Scientific opinion on ergot alkaloids in food and feed. EFSA J. 10, 2798 [158 pp.]. Available at http://www.efsa.europa.eu/de/efsajournal/ pub/2798.pdf Krska, R., Crews, C., 2008. Significance, chemistry and determination of ergot alkaloids: a review. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 25, 722–731. Newman, L.C., Lipton, R.B., 2000. Ergot alkaloids. In: Spencer, P.S., Schaumberg, H.H., Ludolph, A.L. (Eds.),

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Neoptyphodium spp. – Fescue and Perennial Ryegrass Toxicoses Boosinger, T.R., Brendemuehl, J.P., Bransby, D.L., Wright, J.C., Kemppainen, R.J., Kee, D.D., 1995. Prolonged gestation, decreased triiodothyronine concentration, and thyroid gland histomorphologic features in newborn foals of mares grazing Acremonion coenophialum-infected fescue. Am. J. Vet. Res. 56, 66–69. Evans, T.J., 2011. The endocrine disruptive effects of ergopeptine alkaloids on pregnant mares. Vet. Clin. North. Am. Equine. Pract. 27, 165–173. Evans, T.J., Blodgett, D.J., Rottinghaus, G.E., 2012. Fescue toxicosis. In: Gupta, R.C. (Ed.), Veterinary Toxicology, second ed. Academic Press, San Diego, CA, pp. 1166–1180. Johnstone, L.K., Mayhew, I.G., Fletcher, L.R., 2012. Clinical expression of lolitrem B (perennial ryegrass) intoxication in horses. Equine. Vet. J. 44, 304–309. Poppenga, R.H., Mostrom, M.S., Haschek, W.M., Lock, T.F., Buck, W.B., Beasley, V.R., 1984. Mare agalactia, placental thickening, and high foal mortality associated with the grazing of tall fescue. A case report. Am. Assoc. Vet. Lab. Diagn. 27th Annu. Proc. 325–336. Putnam, M.R., Bransby, D.I., Schumacher, J., Boosinger, T.R., Bush, L., Shelby, R.A., Vaughan, J.T., Ball, D., Brendemuehl, J.P., 1991. Effects of the fungal endophyte Acremonium coenophialum in fescue on pregnant mares and foal viability. Am. J. Vet. Res. 52, 2071–2074. Riet-Correa, F., Mendez, M.C., Schild, A.L., Bergamo, P.N., Flores, W.N., 1988. Agalactica, reproductive problems and neonatal mortality in horses associated with the ingestion of Claviceps purpurea. Aust. Vet. J. 65, 192–193. Roberts, C.A., Spiers, D.E., Karr, A.L., Benedict, H.R., Sleper, D.A., Eichen, P.A., West, C.P., Piper, E.L.,

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