Ecosystems and Human Health - Chapter 10 pps

FB1 has been shown to be carcinogenic for animals a promoter and initiator of liver cancer in rats, and there is a high degree of correlation between the incidence of esophageal cancer i

chapter ten

Mycotoxins and other toxins from unicellular organisms

Anything green, that grew out of the mould, Was a wonderful drug to our fathers of old

— Anonymous

Introduction

Mycotoxins are a group of chemically diverse and complex substances present in a wide variety of filamentous fungi (molds) They are secondary metabolites of fungal metabolism of uncertain function Some may have a survival advantage by virtue of their toxicity to competing organisms in the microenvironment The biological function of others is unclear, but many have significant biological activity and several are toxic to mammals They therefore have significant public health and economic implications

It is estimated that 25% of the world's annual food crops are contaminated

by mycotoxins

Some mycotoxins are of interest to pharmacologists and toxicologists because they have served as research tools to study cell function and to identify various types of neurotransmitters and blocking agents Poisonous mushrooms are solid (not filamentous), spore-forming fungi that also con-stitute a health hazard and which have historic, pharmacological signifi-cance, but they are considered separately in Chapter 11

Some human health problems due to mycotoxins

Reports of toxicity from molds are as old as recorded history

Ergotism

The oldest recorded source of fungus poisoning is ergotism Ergot is the common name for the fungus Claviceps purpurpea that affects cereal grains, especially rye, and that produces a number of very potent pharmacologically

active agents that cause toxic reactions when people eat bread made from contaminated flour Periodic epidemics of ergotism have occurred through-out history, and in medieval times these were often attributed to supernatural causes The earliest reference to ergot seems to have been on an Assyrian tablet circa 6000 B.C which refers to “a noxious pustule in the ear of grain.” The Parsees, an ancient religious community in India, referred in their writ-ings to noxious grasses that caused women to abort and to die shortly thereafter The ancient Greeks escaped the scourge of ergotism because they never developed a taste for rye bread, which they referred to by a phrase that translates roughly as “that filthy Macedonian muck.” Because rye bread did not reach Europe until after the decline of the Roman Empire, few if any references to ergotism exist in Roman writings

There was an early association of ergot with St Anthony of Egypt, who lived sometime between 250 and 350 A.D St Anthony is considered to be the founder of Christian monastic life and spent long sojourns in the desert, experiencing visions and hallucinations These frequently involved attacks

by Satan in various guises (wild beasts, soldiers, and women) that either physically attacked him or tempted him Contemporary witnesses claimed that he behaved like an individual being physically abused Because the signs and symptoms of ergot poisoning resembled his attacks, sufferers in the Middle Ages attached religious significance to them and they came to

be known as “St Anthony's Fire” (or sometimes “Holy Fire”) In 1100, the Monastery of the Hospitillers of St Anthony was established at La Motte in France It became the site of pilgrimages by those afflicted with this malady The signs and symptoms included intense burning pain in the extremities followed by a blackened, necrotic appearance; hence the association with fire Epidemics of madness in the Middle Ages may also have been the result

of ergotism

Ergot was employed as an abortifacient long before it was known to be the cause of St Anthony's Fire, and this use continued well into the nine-teenth century when it was finally abandoned because of its highly toxic nature One of the components of ergot is still employed to stop postpartum

hemorrhage because it constricts blood vessels and contracts the uterus To summarize, the symptoms of ergotism include drowsiness, nausea, vomit-ing, muscle twitch, staggervomit-ing, gangrene, hallucinations, and abortion Ergot contains a veritable potpourri of pharmacological agents that were charac-terized by Sir Henry Dale in the early 1900s and contributed to the devel-opment of many new drugs

The active components of ergot, the ergot alkaloids, are all derivatives

of lysergic acid They bear some molecular resemblance to adrenaline (epi-nephrine), dopamine, and serotonin — all central neurotransmitters Meth-ysergide is the precursor of LSD Two derivatives still have medicinal appli-cation Ergometrine (ergonovine) is the one used to control postpartum

bleeding Ergotamine is a powerful vasoconstrictor responsible for the gan-grene of ergotism In very small doses it can be used to treat migrane

Ergotism also affects farm livestock, causing similar signs and, in addition, loss of milk production in cattle and necrotic combs, feet, and beaks

in poultry

Aleukia

Aleukia (literally “absence of white cells”) occurs when millet and other grains that have become moldy are consumed The toxin is a tricothecene from species of Fusarium and it damages bone marrow Because of severe food shortages in Russia during World War II, the eating of moldy grain resulted in several epidemics of aleukia, including a very large one in 1944 The mortality rate of those with marrow damage was 60% Other symptoms included hemorrhages in the skin and mucous membranes The condition

is also referred to as alimentary toxic aleukia

Aflatoxins

Aflatoxins are a family of heterocyclic, oxygen-containing compounds secreted by the molds Aspergillus flavus and A parasiticus These molds grow abundantly on many kinds of plants in very hot conditions Peanuts are especially prone to infection Taste is not affected and the toxins are heat stable Aflatoxin B1 (AFB1) is the most frequently encountered member of the group and it is a potent carcinogen in experimental animals (rodents, birds, and fish) Rats fed a diet containing 15 ppb AFB1 develop hepatitis, which is often followed by cancer of the liver Epidemiological studies of populations in Uganda, Kenya, and Thailand have shown a close correlation between the incidence of liver cancer and the consumption of food contain-ing aflatoxins It is estimated that the risk factor for liver cancer is increased

10 times by AFB1 Infectious hepatitis also increases the risk by a factor of 10

If present together, the risk is increased 100-fold Other aflatoxins include

B2, G1, and G2 Lactating cattle excrete hydroxylated metabolites M1 and M2

in milk These have lower toxicity than the parent compounds but are of concern because of the large amount of milk consumed by infants and young children

Thus, this natural carcinogen, at least as potent as TCDD in animal studies, is strongly implicated as a cause of human cancer, unlike the latter synthetic agent, that has received much more media coverage Experiments employing human liver microsomal preparations indicate that cytochrome P450 (Cyp) 3A4 is largely responsible for the formation from AFB1 of the 8,9-endo-epoxide, a genotoxic metabolite Cyp 1A2 also forms this epoxide but is much less active It forms other, nongenotoxic metabolites and thus may play a role in detoxification An inhibitor of Cyp 1A2, alpha-naphthofla-vone, increased the formation of the 8,9-epoxide The exo-epoxide of AFB1 (i.e., preformed before ingestion) is detoxified by conjugation with glu-tathione Experimental evidence suggests that species differences in toxicity are due to differences in the rate and extent of conjugation with glutathione

Whereas rat liver cytosol conjugated endo-epoxide, mouse liver cytosol con-jugated the exo-epoxide almost exclusively and both were much more active than the cytosol of human liver cells The mucus cells of the intestine also make the 8,9-epoxide, but because they are sloughed frequently, this does not constitute a risk factor for cancer

Other aflatoxins found together are B2, G1, and G2 Acute toxicity also can occur in humans In 1974, an outbreak in India resulted in about 100 deaths, and in Kenya 12 died in an outbreak in the early 1980s

The aflatoxin story began in 1960 with a serious and mysterious outbreak

of a disease in turkeys in Great Britain Turkey poults developed loss of appetite, feeble fluttering, lethargy, and frequently died within a few days Necropsies revealed hemorrhage and necrosis of the liver and kidney The disease was dubbed “X” disease Outbreaks also occurred in ducklings in Europe, the United Kingdom, and Africa The common denominator was groundnut (peanut) meal used as feed and subsequently shown to be con-taminated with A flavus Livestock (mammals) may also be affected Death has occurred in humans consuming an estimated 6 mg/day of aflatoxin B1

In North America, there is concern about the long-term effects of con-suming low levels of aflatoxins and monitoring systems are in place Fortu-nately, the mold is not adapted to the colder northern climate so that Cana-dian-grown peanuts are free of the toxins The majority of peanuts and peanut butter are still imported from subtropical climes, however Peanuts are not the only source of these toxins Because of drought conditions in the United States in the summer of 1988, up to 30% of the corn crop may have been contaminated The Quaker Oats Co was turning away almost one truckload in five at its Cedar Rapids (Iowa) plant in the fall of 1988 The company tests six samples from each truck Inspections of the 340,000 tons

of corn crossing the Canada–U.S border annually were stepped up in 1988–1989 by Agriculture Canada The general structure of aflatoxins is shown in Figure 37

Fumonisins

Produced by Fusarium monilforme and F proliferatum, these mycotoxins are ubiquitous in many parts of the world, including South Africa where they were first identified and many states bordering the Great Lakes All of the fumonisins (FB1, FB2, FB3) are potentially carcinogenic Many experts feel that it will become the most significant mycotoxin for human health FB1 has been shown to be carcinogenic for animals (a promoter and initiator of liver cancer in rats), and there is a high degree of correlation between the incidence

of esophageal cancer in humans and the presence of FB1 in corn in specific areas of South Africa The fungus infects corn, millet, sorghum, and rice around the world Fumonisins are toxic for many species of animals, espe-cially horses, and outbreaks have caused numerous deaths in horses and

swine in Texas, Iowa, and Arizona Severely infected corncobs may contain

up to 900 mg/kg Symptomatology in various species is as folllows:

• Swine: vomiting, convulsions, sudden death, abortion, pulmonary edema (porcine pulmonary edema syndrome) Symptoms may occur at levels above 20 to 50 mg/kg of FB1

• Poultry: ataxia, paralysis, sudden death, stunted growth Toxicosis occurs at levels of contamination of 10 to 25 mg/kg of FB1

• Cattle: poor weight gain, liver damage

• Horses: equine leukoencephalomalacia (ELEM), brain degenera-tion with focal necrosis, blindness, wild behavior, liver damage, staggering, ataxia Symptoms may occur at levels of 10 mg FB1/kg over 40 days

FB1, FB2, and hydrolyzed FB1 have been shown to be specific inhibitors

of de novo sphingolipid synthesis and sphingolipid turnover FB1 has been shown to inhibit sphingosine (sphinganine) N-acetyltransferase, leading to the accumulation of sphyngoid bases This has been shown to stimulate DNA synthesis, and it is hypothesized that this interference with normal cell function could account for the toxicity of fumonisins

The fumonisins are themselves analogs of sphingosine, with structural similarities to the phorbol esters, which are known carcinogens (see Chapter 11) The general structure of fumonisins is shown in Figure 37

Figure 37 Chemical structures of aflatoxins and fumonisins.

O

O AFB 1

R 2

R 1

CH 3

NH 2

CH 3

CH 3

OH AFB 1 8,9-Epoxide

O

CH 3 O

O

O O O

CH 3 O

Fumonisin B 1 : R 1 = 0-PTCA*, R 2 = R 3 = OH

Fumonisin B 2 : R 1 = 0-PTCA, R 2 = H, R 3 = OH

*PTCA = Propanetricarboxylic acid

Other mycotoxic hazards to human health

Several mycotoxins may be potential health hazards by virtue of direct toxic effects or because of carcinogenic or teratogenic properties, although it must

be emphasized that hard evidence linking these to human health problems

is scanty Ochratoxin A is formed by Aspergillus and Penicillium species It has been shown to be embryotoxic and teratogenic in several laboratory species of mammals (pig, dog, mouse, rat) and birds and is therefore viewed

as a potential human teratogen Acute effects in animals (including swine and poultry) include renal and hepatic destruction Both humoral and cel-lular immune systems are adversely affected Contamination of bread and cereals has been documented in parts of central Europe (Yugoslavia, Bul-garia, Poland, and Germany) and levels have been detected in human milk, urine, blood, and kidneys

Patulin is potentially a carcinogenic toxin produced by several species

of fungi, including some Penicillium spp It is a highly potent inhibitor of RNA polymerase, having a strong affinity for sulfhydryl groups It therefore inhibits many enzymes Teratogenicity has not been demonstrated in mam-mals but embryolethality occurs at higher doses (2 mg/kg i.p.) A common source of patulin is Penicillium expansum, a common spoilage microorganism

in apples Apple juice can sometimes contain significant amounts of patulin Acute toxicity in rodents is manifested primarily as gastrointestinal symp-toms, including hemorrhage Carcinogenicity studies in rats were negative, but clastogenic activity has been shown in some systems

T-2 toxin is produced by various Fusaria and is both potent and common

It inhibits protein and DNA synthesis and is therefore potentially teratogenic and carcinogenic Acute effects include loss of appetite and vomiting Fungi tend to produce mixtures of toxins so that exposure to a single agent is unlikely to occur There is some experimental evidence that ochra-toxin A potentiates the teratogenic effects of T-2 It must be emphasized that any mycotoxin that has been shown to be toxic in several mammalian species (including various farm livestock) must be regarded as potentially toxic for humans

Economic impact of mycotoxins

In addition to their direct effects on human health, mycotoxins have a tre-mendous impact on agriculture Through spoilage, field crops are rendered useless for animal or human consumption (the loss of 20% of the corn crop noted above is one example) Poor weight gain and outright illness occur in livestock that consume contaminated feeds Losses are difficult to estimate but they undoubtedly run to many millions and possibly billions of dollars

in North America The presence of trace quantities in meat, dairy products, and eggs constitutes a further, if largely unconfirmed, health hazard to people In the Great Lakes basin, various species of Fusarium are the most

common offenders, especially in eastern Canada They may produce a host

of toxins with potent pharmacological actions

Fusarium life cycle

Fusarium graminearum is the fungus responsible for maize ear rot in corn and head blight in wheat Its life cycle is typical of all Fusaria Spores survive in crop debris from the previous season (stubble, stalks and seeds) to re-infect the next year's crop Intensive farming practices that involve planting suscep-tible species in the same fields year after year thus favor the spread of infec-tion Birds such as starlings and red-winged blackbirds, which puncture the corn kernels to eat the milk, may also spread spores Insects such as the picnic

or corn-sap beetle seek out damaged kernels and also may spread spores Certain weather conditions favor the spread of infection Fungus growth

is favored by warmth (15 to 35°C) and by surface wetness for more than

48 hr After the infection is established, weather is not critical to the produc-tion of the toxin Mold growth will continue throughout the season, and even afterward if not properly dried or if storage conditions are poor (too damp, too warm, poor air circulation) Mold growth can even occur during feed preparation and in poorly cleaned feed troughs Late harvest may allow the growth of another fungus, F sporotrichiodes, which produces the toxins T-2, HT-2, and diacetoxyscirpenol

Contaminated grains thus will contain complex mixtures of toxins and metabolites The following is a list of the other important ones and their effects

Trichothecenes

Zearalonone

This estrogen-like toxin causes (in swine) swollen, red vulva, vaginal and rectal prolapse, vulval enlargement in piglets, and fertility problems Devel-opmental defects and lethality have been shown in some laboratory species

Vomitoxin (deoxynivalenol or DON)

This trichothecene causes decreased feed intake and reduced weight gain

in pigs at about 2 mg/kg of feed, vomiting, and refusal of feed at very high concentrations (>20 mg/kg feed) DON will be used as a “prototype” mycotoxin to illustrate agricultural problems associated with these agents (see below)

Species differences in DON toxicokinetics

Swine appear to be much more sensitive to the anorexic and weight loss effects of DON than ruminants (cattle, sheep) or poultry, which are very

tolerant In one study, laying hens actually preferred a diet containing 5 ppm DON in preference to clean feed These differences are due, in part, to differences in absorption and, in part, to differences in biotransformation and elimination Studies with radiolabeled DON indicated that sheep absorbed 9% or less of an orally administered dose, in turkeys 20% or less was absorbed, whereas pigs absorbed up to 85% of a single oral dose Intravenous administration of radiolabeled DON in sheep revealed an initial distribution phase (t1/2 = 18 min) followed by an elimination phase (t1/2 = 66 min) A glucuronide conjugate was formed and comprised 15 to 20% of plasma levels In turkeys there was an extremely rapid distribution phase (t1/2 = 3.6 min), a rapid elimination phase (t1/2 = 46 min), and the formation of a conjugate (probably glucuronide) comprising up to 10% of the total dose Again, swine showed a much different picture There was a very rapid distribution phase (t1/2 = 5.8 min), a secondary slower distribution phase (t1/2 = 96.7 min), and a very prolonged terminal elimination phase (up

to 510 min) There was no evidence of significant biotransformation in swine Thus, it took 7 to 10 times longer for swine to clear the toxin than for the other two species

Toxicity is a function of many factors: the concentration of toxin reaching the target organ (which in turn is affected by the rate of absorption at the portal of entry), the extent of distribution to non-target sites (i.e., where no toxic effects occur), the rate and extent of biotransformation to nontoxic metabolites (or to toxic ones as the case may be), and the rate of elimination

in urine and feces The effect of species differences in some of these factors was introduced in Chapter 2 Volume of distribution (Vd) and clearance data provide some information regarding the fate of the absorbed toxin The apparent Vd is a mathematical calculation of the volume of diluent required

to dilute an administered dose of a substance (usually intravenously) to the observed concentration Vd = M/C where M = mass (amount of substance) and C = concentration of substance Calculations of Vd for DON yielded values of 0.167 L/kg for sheep vs 1.3 L/kg for swine, suggesting that in the former DON was confined mainly to the extracellular fluid, whereas in the latter it was taken up by tissues Initial systemic clearances were not all that different, being 1.37 mL/min/kg for sheep and 1.81 mL/min/kg for swine

An interpretation of this data suggests that DON is initially rapidly distributed

to tissues and then slowly released back into the plasma, yielding the slow, terminal elimination phase Turkeys also had a very large Vd (2.33 L/kg), but they also had an extremely rapid clearance (35.0 mL/min/kg), indicating that DON was rapidly distributed to tissue compartments but not held there Thus, the extreme sensitivity of swine to DON is the result of:

• High oral bioavailability

• Wide distribution to tissues

• Slow elimination from the body

• Minimal detoxification through biotransformation

Other trichothecenes

T-2 and HT-2 toxins and diacetoxyscirpenol are more toxic than DON and cause reduced feed intake, vomiting, irritation of the skin and gastrointesti-nal tract, neurotoxicity, teratogenicity, impaired immune function, and hem-orrhage Adverse effects seen in farm animals are generally caused by mix-tures of these toxins rather than by a single toxin Blending of several grains

in the preparation of feed may further contribute to the toxic diversity of the mixture Potentiation of effects may occur Thus, DON at the subthresh-old level of 1 mg/kg plus low (ppb) concentrations of T-2 and other uniden-tified toxins may cause severe toxic manifestations in a sensitive species such

as swine The chemical structures of some of these toxins are shown in

Figure 38 Chemical structures of several mycotoxins.

HO

OH

OH

OH

CH3

CH 3

CH 3

R 3

R 2

OH

CH 2

R 1

CH3 CH3

CH2OH

O O

O

O O O

H

Zearalenone

O O

Deoxynivalenol ( DON, vomitoxin )

T - 2 : CH 3 COO at R 1 and R 2 , ( CH 3 ) 2 CHCH 2 COO at R 3

HT - 2 : OH at R 1 , CH 3 COO at R 2 , (CH 3 ) 2 CHCH 2 COO at R 3

Diacetoxyscirpenol ( DAS ) : CH 3 COO at R 1 and R 2 , H at R 3

Detoxification of grains

Because of the diverse chemical properties of the mycotoxins, physical and chemical procedures that are effective against one toxin may have little or

no influence on the toxicity of others Thus, there is no single process that can be used The most important control factor must be the avoidance of conditions favoring fungal growth at all stages of food production

Harvesting and milling

Infected kernels may represent less than 5% of all the grain They may be broken or shriveled and in wheat may take on a “tombstone” appearance

In corn, the tips of cobs may have shriveled, highly infected kernels con-taining up to 3000 mg/kg of DON Grain dust may be very contaminated Screening and blowing will remove much of the dust, particles, and with-ered kernels

Wet milling of corn has been shown to remove about two thirds of the T-2 toxin, but milling had little effect on the DON content of flour from hard wheat, nor did baking the flour into bread Some milling procedures may actually increase the DON content of the finished product In mild infections, washing and roasting may significantly reduce toxin levels

Chemical treatments

Laboratory tests have shown that moist ozone, ammonia, microwaving, and convection heating reduce DON concentrations in moldy grain Aqueous sodium bisulfite plus heat effected a complete detoxification Studies have shown that this technique resulted in normal feed intake and weight gains when contaminated corn was treated and fed to swine

Binding agents

The addition of binding agents, such as bentonite, anionic and cationic resins, and vermiculite-hydrobiotite were tested on the toxicity of T-2 in rats Ben-tonite prevented T-2 toxicosis by blocking intestinal absorption Polyvi-nylpyrrolidone or ammonium carbonate had no effect on DON toxicity in swine Alfalfa fiber has been shown to partially overcome the growth-depressing effect of zearalenone in rats but not the estrogenic effects in swine

Other techniques

Dilution of contaminated feed with clean feed will improve palatability and feed consumption More concentrated diets with respect to calories, protein, etc may overcome the effects of a moderate reduction in feed intake Exper-imentally, antibodies against zearalenone have been raised in swine and shown promise in protecting against its toxic effects