Zearalenone Mycotoxin

Photo of Sharol Tilgner
Zearalenone structure

What Is Zearalenone Mycotoxin

Zearalenone (ZEN),  is a mycotoxin (fungal toxin) produced by several fungi of the Fusarium genus, including Fusarium graminearum (Gibberella zeae), F. culmorum, F. cerealis, F. equiseti, and F. semitectum.  It is primarily produced by F. graminearum and F. semitectum. Zearalenone is a macrocyclic Beta-resorcyclic acid lactone and has a negative charge. It's claim to infamy lies in the fact that it is a known endocrine disruptor, specifically enhancing estrogenic effects.

Zearalenone is abbreviated as are most mycotoxins with long names. However, it is abbreviated as ZEN and ZEA both in various research articles making it confusing.

Where Is Zearalenone Mycotoxin Found

Zearalenone is found in food but may also be found in air borne dust in buildings where mold contaminated feed exists, such as in the air of poultry buildings or facilities processing mold contaminated grains into flour.

Common Foods:

Zearalenone is found in corn, wheat, rice, oats, barley, sorghum and rye as well as grain flours or processed food made from them. It may additionally be found in moldy hay as well as malt, soybeans and beer. Yes, not only does the hops in the beer cause estrogenic effects, but if there is zearalenone (ZEN) in the malt or grain used to make your beer you also have the estrogenic effects from ZEN.

In the USA and Canada zearalenone is most frequently found in corn and wheat, while in Europe is often found in  wheat, rye and oats.

Dairy, Meat And Eggs May Contain Zearalenone Mycotoxin Or Metabolites

Zearalenone or metabolites of ZEN may also be found in milk since it has been shown to pass through animals milk to the nursing young. It is used in the agricultural industry (as opposed to family farmers) as a growth hormone due to its estrogenic effect. Therefore it may be found in the animal meat or in milk from these animals. Zearalenone may also be found in eggs.

You might think that meat eaters would have higher exposure to ZEN but according to the European Food Safety Authority, vegetarians have twice the the exposure than non-vegetarians. There is also a concern that children with celiac who have higher exposure to corn products will also have higher exposure to ZEN and its modified metabolites. It is thought that exposure to ZEN through food of animal origin is relatively low while plant origin is much higher. However, in my opinion there is not enough research on this matter to know that. There are cases of ZEN exposure from animals sources such as the Olympian runner's ZEN exposure from hamburgers that I describe in this article.

Preventing Zearalenone Mycotoxin Exposure

Regulation Of Zearalenone

Several nations have established guidance levels or maximum permissible levels for ZEN in foods, ranging from 50 to 1000 μg/kg
(ppb). The United States does not regulate ZEN. There are guidelines for feeding agricultural animals but no enforceable regulations on allowable limits in food for people or animals.

Dietary exposure estimates suggest that current exposure levels to ZEN may be close to the tolerable daily intake for ZEN as established by the European Food Safety Authority, which as of 2011 are 0.25 μg/kg body weight. The tolerable intake of the metabolites for ZEN are not known at this time as far as I can tell and some of them are more harmful than ZEN itself (discussed below). For more information on metabolites, see the section below on "Masked Mycotoxins".

The Role Of The Farmer

Zearalenone (ZEN) is not visible to the eye, so it can be consumed by unaware humans. The farmer is key in preventing Fusarium (the mold that makes ZEN) growth during farming, as well as following good harvesting methods. Farming practices such as supporting the soils healthy microbial/fungal/parasitic biome, rotating crops, and supplying nutrients to the soil, will all go a long way to supporting a healthy plant, that will do better in droughts, and not attract insects (insects damage plants allowing for fungal entry into plant). This helps to decrease fungal growth and mycotoxin production. Keeping the plants healthy and happy will decrease plant stress, and decrease the plants susceptibility to all disease, including fungal disease. It has been noted that the stress of drought (especially during grain formation), and insects will increase mycotoxins, including ZEN on plants. Additionally, rainy periods at the end of flowering, or high moisture during the late preharvest period will increase the chance of ZEN and other mycotoxins. Any physical damage to the growing plant that opens its protective surface can allow fungus to penetrate into the plant more easily. Areas of the world such as Italy where there are warm, and humid summers are  more likely to grow Fusarium spp. mold that may make ZEN.

Farmers can decrease mycotoxin infestation including Fusarium spp. that make ZEN by caring for the soil and supplying nutrients necessary. The application of lime and manure/compost have been shown to be effective in reducing A. flavus contamination as well as aflatoxin levels by 50-90%. Lime contains calcium that helps thicken the cell walls of the plants to protect them, and the use of farm yard manure/compost facilitates growth of microorganisms that suppress soil infections.

The Role Of Processing And Storage

High humidity and low temperatures have been shown to support the production of zearalenone by Fusarium graminearum in corn. Corn and wheat are most affected by this mycotoxin and a high relative humidity during storage favors production of ZEN.

Good  processing as well as storage and and transport practices help to decrease mycotoxin formation on plants.  All known contaminated food needs to be removed from the market. However, in countries where food may be less availble or if a farmer/processor is not honorable, mycotoxin contaminated food may end up on the market. Broken and damaged grain kernels usually contain most of mycotoxin contamination although they constitute only 3–6 % of the bulk food. Removing kernels with extensive mold growth, broken kernels, and fine materials such as dirt and debris can be achieved by sieve cleaning, which significantly lowers total mycotoxin contamination.

After harvest contamination can lead to throwing the food out, or taking human food and giving it to animals. When contaminated food is given to animals, chemicals to decontaminate the product, or adsorbants, or even enterosobents are given with the feed being fed to animals. This lessens the mycotoxin load, but not completely.

There is processing that can lower mycotoxin contamination of food. However, some activities can actually release metabolites from "masked" mycotoxins also. There are enzymes that are known to decrease certain myctoxins. Methods that have been shown to reduce general mycotoxin contamination are cleaning; milling; brewing; fermentation; cooking; baking; frying; roasting; flaking; alkaline cooking; nixtamalization, hulling of grains and extrusion. Concentrations of some mycotoxins can be reduced substantially while others, such as deoxynivalenol (a trichothecene also made by Fusarium and often found with ZEN), are relatively resistant to degradation. Zearalenone is known for being fairly stable under normal cooking temeratures except under alkaline conditions, or during heating under a high degree of pressure. High temperatures can partially eliminate it. Milling has been shown to lower ZEN content. Traditional stone milling has been shown to remove much more ZEN from wheat than the modern roller milling process.  Dehulling of grains appears to reduce the amount of ZEN. Fermentation has also been shown to lower ZEN levels as it often does with mycotoxins. Exposure to UV light has also shown degradation of ZEN.

Additional Combinations Of Food And Other Mycotoxins

There is the additional factor of how ZEN and other mycotoxins interact in the body as well as other substances in the food. There are additional endocrine disruptors that may be in the food and we have no idea how any of them interact together. The co-exposure of mycotoxins in human and animal food is known to take place, however we have little data on how they interact and if these combinations cause increased or decreased toxicity. We might also want to know how ZEN interacts with other estrogenic phytoestrogens such as the ubiquitous soy? Soy can grow Fusarium species and therefore be contaminated with ZEN, but it also has an estrogenic effect of it's own.

Not Regulated, But There Are Suggested Levels

The USA has given farmers suggestions of levels of ZEN that are okay in grains, and it is up to farmers to have good growing practices and manufacturers to have good manufacturing practices to keep the levels low. However, there is little testing done, and that which is done is not know to the public unless you look for it as I have done. This works great as long as eveyrone has good intetions, however there are some issues that can arise. For instance, there is no one making sure a corn batch that is tested as high does not end up in your dogs food. I would suggest you make your own dog food anyway, just as you should be making your own food from scratch.

Zearalenone Mycotoxin Levels In Recent Corn Harvests

In 2018 there was late-season wet weather which is known to increase ZEN levels. In 102 samples collected by the Iowa Department of Agriculture and Land Stewardship (IDALS), the levels of zearalenone were detectable in 82 samples and deoxynivalenol (also called vomitoxin and also made by Fusarium species) was detetable in all samples. These were samples from all regions of Iowa where a lot of corn is grown for agricultural purposes. Neither of these mycotoxins are regulated by the FDA, but there are suggestive feeding limits for some animals. The zearalenone was found to be 54% above USA suggested feeding limits for sensitive livestock animals (pigs - young pigs) and the deoxynivalenol was 48% above suggested feeding limits of sensitive livestock animals. Corn that has been used for distillation into alcohol is often used as a feed for animals, and it was noted that the processing concentrates toxins to 3 times their usual amount in these distillers grains, making any distillers grains from this corn that is already high in toxins, even more toxic. The question is where did all this corn go? I could not find an answer to that. It is not illegal to sell it.

I found additional sampling surveys undertaken by the company Biomin (they sell solutions for farmers with contaminated feed, so they are not impartial) for 23 states examining 6 mycotoxins including ZEN in 2018, and the end of the year 2019 sampling will come out soon. There is only preliminary data so far. The results are  alarming to me and you can find the current data for 2018 here.

Anyone looking at this data would conclude that we should be taking a closer look at food contamination, not just for livestock and pets, but for humans too. It might be helpful to also have some legal limits of how much contamination can exist and still sell the grain. This would most likely raise the cost of grain, pet food, meat, eggs and milk which is an issue for those that make these decisions. There is also the issue of not knowing all the metabolites that exist when the "mother mycotoxin" such as ZEN is broken down. We don't really know what we should be looking for in most of these cases. There could be metabolites of the mother mycotoxins such as Zearalenone that are unknown that we can't even begin to test for. They may even be more toxic than the mother mycotoxin. Further on in this article, I will explain the role of ZEN metabolites. For know, just know that ZEN breaks down into other metabolites both in the plants, meat you eat as well as your own body. Some of these metabolites are safer and some more toxic. The conjugated forms are considered less toxic.

We have already identified some that are more estrogenic than ZEN. Some of the major metabolites that are currently known are the phase I metabolites called alpha and beta-zearalenol (alpha and beta-ZEL), alpha and beta-zearalanol (alpha and beta-ZAL), zearalanone (ZAN), as well as the phase II metabolites (conjugates) which are largely glucuoronides, glucosides and sulfates. However, over 30 modified forms of ZEN have been identified so far and who knows how many more there are and we don't have availble testing for them at most labs and think of the cost.

Zearalenone And Masked Mycotoxins In Food

Conjugates of fusarium mycotoxins are found in foods, and are called "Masked toxins" as they are not generally tested for, and therefore not known they are in the food. Technically, conjugates are much safer than the other forms, but the issue is that conjugates can be transformed back into the other forms of ZEN metabolites that are much more toxic. The conjugates are formed by the plant as it conjugates mycotoxins to polar groups. Plants have their own phase II conjugation activities just as animals do. Conjugates commonly occur along with their parent compounds in food and animal feed. The conjugates deoxynivalenol-3-glucoside (D3G) and zearalenone-14-glucoside (Z14G) are the most common masked mycotoxins found in foods when tested. Deoxynivalenol (a trichothecene) and Zearalenone are often found together , made from the same mold called Fusarium spp. These compounds, Deoxynivalenol-3-glucoside (D3G) and zearalenone-14-glucoside (Z14G) could be toxic themselves, or they could be hydrolyzed to release the parent aglycone after ingestion causing increased exposure to mycotoxins that is not accounted for with conventional lab analysis since testing of food does not consider conjugates. Specific intestinal bacteria have been shown to convert D3G into DON, so this is a highly likely possibility. For this reason, masked mycotoxins should be considered when evaluating population exposure to mycotoxins in food. I will discuss both deoxynivalenol and zearalenone since they are often made together by Fusarium spp. molds. You could end up with a mycotoxin toxicity representing both of them and it would be good to know you could have two different mycotoxins creating a mixed health picture.

DON absorption takes place mainly in the duodenum and in the small intestine. DON causes impairment of gut motility and appetite. Effects of DON are associated with neuroendocrine signaling, proinflammatory gene induction, disruption of the growth hormone axis and altered gut integrity. It appears to involve altered neuroendocrine signaling at both the enteric (gut) and central levels. It is implicated in reproduction disorders of animals and hyperestrogenic syndromes in humans. Elevation of serotonin levels in the gut could be related to the peripheral and central serotonergic effects of DON.

ZEN's metabolites Z14G and Z14S are effectively deconjugated by the human colonic microbiota (the flora make beta-glucuronidase-the deconjugating enzyme), releasing their toxic aglycones and generating yet unidentified catabolites. The aglycone is much less polar than Z14G and Z14S and is more readily absorbed. Additionally intestinal mucosal cells may convert ZEN into the more estrogenic Phase I metabolite alpha zearalenol/zearalanol.

The average rates of DON glucuronidation were 76%, and there was 68% excretion in a human volunteer's urine 3-10 hours after exposure. The investigation of formed glucuronides revealed DON-15-glucuronide as main conjugation product besides DON-3-glucuronide. Recently, for the first time in human urine a third DON-glucuronide was detected and the fate of ingested masked DON forms (3-acetyl-DON and DON-3-glucoside) was preliminary assessed.

Health Effects of Zearalenone Mycotoxin

Acute Toxicity

Zearalenone is currently thought to exhibit low acute toxicity, although research does show it exists.

Long-term exposure to ZEN is considered to pose a health risk due to its potent estrogenic activity.

Chronic Toxicity

Chronic toxicity encompasses organ damage/body system damage as well as endocrine disruption. We know the most about it's role as an endocrine disruptor due to the issues that have arisen in animal rearing.

An Endocrine Disruptor

Although ZEN has a non-steroidal structure, it does fold upon itself in such a manner that it can bind to both estrogen receptor beta, and alpha, thereby causing morphological and functional effects on the reproductive system. It has been shown to produce reproductive disorders in animals and humans.

Zearalenone (ZEN) mimics the reproductive hormone estrogen. Zearalenone and its metabolites have been shown to compete effectively with specific binding sites of the estrogen receptors. Zearalenone acts by binding estrogen receptors (ERs), as an agonist to ER-alpha and a mixed agonist/antiagonist activity to ER-beta. The affect of ZEN will depend on the sex of the person or animal and if they are prepubertal, menstruating, pregnant, or menopausal as well as how well they hydroxylate, conjugugate and de-conjugate it. In Puerto Rico, zearalenone was found in the blood of children with precocious sexual development exposed to contaminated food.

There is also some data available showing it may have an affect on aromatase.

Increased Estrogenic Potency of Zearalenone Mycotoxin From Some Metabolites

The biotransformation process (detox process) and metabolites formed  can increase estrogenic potency of zearalenone.

The phase one biotransformation called hydroxylation converts zearalenone into the metabolites, alpha-zearalenol and beta-zearalenol (alpha and beta-ZEL) as well as alpha-zearalanol and beta- zearalanol (alpha and beta-ZAL). Alpha-hydroxylation to alpha-zearalenol/zeaeralanol results in an increase of estrogenic potency compared to zearalenone, while beta-hydroxylation into beta-zearalenol/zeaeralanol results in less estrogenic capacity. Animal species differ in hydroxylation capacity and therefore there is a difference of sensitivity towards ZEN intoxication. Phase II biotransformation is to a lesser toxic structure as is usually true.

Possible Signs Of Hyperestrogenism From Zearalenone Or It's Metabolites
  • vulvar edema
  • breast edema
  • vulvo-vaginitis
  • enlargement of the uterus
  • ovarian cysts
  • impaired maturation of ovarian eggs
  • enlarged uterus
  • ovaraian atrophy
  • endometrial changes
  • vaginal prolapse
  • decreased fertility
  • increased embryonic and fetal death
  • abortion
Organ Damage Due To Zearalenone Mycotoxin

Zearalenone studies are largely in animals although there are some human studies. They have shown damage to reproductive organs, the intestines, the immune system, liver, kidney and spleen. In animals it can induce an enlarged uterus, ovaraian atrophy, endometrial changes, vaginal prolapse, swelling of the vulva and vagina (known as vulvovaginitis), enlarged breast glands, anestrus (periods of infertility), decreased fertility, increased embryonic and fetal death, and abortion. Additionally ZEN is associated wtih reduced litter sizes in animals and changed weights of adrenal, thyroid and pituitary glands in offspring as well as changes in serum levels of progesterone and estradiol.

 

Other Effects

Zearalenone can causes increases and decreases in inflammatory cytokines and it may be organ dependant according to some studies. It has been shown to increase TNF-alpha, IL-8,IL-6, IL-1beta in the spleen. There was significant increase in the powerful inflammatory marker TNF-alfpha in this study. The same researchers looked at liver activity of inflammatory cytokines and found there was a decrease for all pro and anti-inflammatory cytokines when affected by ZEN. This supports a prior researcher hypothesis that ZEN has different affects on cytokines in the liver and spleen.

 

Zearalenone Metabolite Used As An Animal Feed Additive To Increase Growth

The metabolite α‐zearanol, one of the phase I metabolites of ZEN, is used as a growth promoter in non‐European Union countries under the name of Zeranol (trade name Ralgro®). It is banned in Europe, but commercially available in many other countries, and used as a growth hormone to increase weight gain in beef cattle due to it's estrogenic effect. I was looking at the ralgo.com website recently and saw on their home page that they were selling this product as a method to conserve land, and water resources, and leave something for the next generation. This product is being used to fatten up the animals, and they are presenting it as a way to use less feed, which saves crop land, less pasture land and uses less water. Certainly if you can use an endocrine disrupting mycotoxin to fatten up your animals faster than normal, it will save use of feed which ultimately does save on use of pasture land, crop land and water. Is this how we want to preserve our resources though? This is the problem that arises when we as consumers only hear what we want to hear, such as using a product to preserve the water, land or decreasing carbon units. Certainly this product will do all of these things. However, none of these things in themselves means anything in reality. They are just methods to manipulate an uneducated public. We really have to look at each individual situation rather than relying on soundbits to make decisions. Real life is always multifaceted and every situation is unique. It is quite disheartening that this mycotoxin would actually be fed to animals to fatten them. Then people, or pets, or other farm animals eat the meat, milk, or eggs from those animals. Excretion of ZEN and it's metabolites in meat, milk and eggs has been documented. Zeranol and its metabolites present in the meat of zeranol-implanted beef cattle may indeed be considered an endocrine disruptor for human consumers. In human females who eat food contaminated with ZEN, studies have associated it with female reproductive changes such as breast cancer and precocious puberty.

You may remember in 2017 that Ajee' Wilson, the 2006 Olympian tested positive for zeranol and was accused of taking the anabolic agent. After reviewing her food purchases, it was decided that the zeranol was from contaminated American beef that she had eaten.

 

Mechanism Of Injury

Zearalenone can activate the pregnane X receptor and thereby increase the transcription of many genes, including Cytochrome P 450 enzymes.

Zearalenone has been found to affect genes by both up-reguatlion of some genes and down-regulation of others.  One study found 480 genes altered significantly with 60% up-regulated and 40% down-regulated. The largest group of genes affected were involved in cellular signalling pathway (46%), cytokine network(13%) and the inflammatory response (10%). In the spleen there was an increase in the expression and synthesis of pro-inflammatory cytokines while in the liver there was a dramatic decrease in pro- and anti-inflammatory cytokines as well as signaling molecules p-38MAPK and NF-kB.

Zearalenone and its metabolites bind to estrogen receptors and are agonists/antagonists. The estrogenic activity of ZEN and its metabolites has been directly correlated with their binding affinity to estrogen receptors estrogen receptor-α and estrogen-β . it is thought to be an agonist toward ER-alpha while being a mixed antagonist/ agonist to ER-beta. The affinity of ZEN is higher for estrogen receptor-α. The estrogen receptors are expressed in manay organ tissues including the uterus, breast glands, bone, brain, as well as on the surface of the immune system cells (estrogen receptor-α is found on T cells, NK cells, macrophages, Estrogen receptor-β is found on B cells and monocytes) which could become a possible target for zearalenone or metabolites of zearalenone.

Zearalenone and its metabolites produce a syndrome called hyperestrogenism.  Alpha-zearalenol is the most estrogenic. In a rat study ZEN metabolites were found to be more or less estrogenic in this order: α-zearalenol > α-zearalanol > zearalenone > zearalanone > β-zearalanol > β-zearalenol.

It has been demonstrated in vitro that zeranol (used to pump up beef cattle- see above) enhances cell proliferation and increases the ERα content of ER-positive breast cancer cells. Research has shown that exposure to low doses of zeranol may increase the risk of breast cancer by increasing estrogen levels in adipose tissue through upregulation of aromatase mRNA expression and aromatase activity in preadipocytes. In vivo zeranol may stimulate estrogen release, thus inducing the excessive proliferation of breast cells.

Zearalenone has been shown to alter the normal microecology by inhibiting the growth of beneficial bacteria in the intestine of animals. Mycotoxins have been shown to be related to intestinal inflammatory diseases in humans. Several mycotoxins have been found to induce intestinal alterations that are similar to those seen during the progression of inflammatory bowel diseases. The data leads us to suspect a role of all food-associated mycotoxins in the induction and/or persistence of human chronic intestinal inflammatory diseases in genetically predisposed patients. Indeed, mycotoxins that end up in the digestive tract will be absorbed by the small intestine, and it is the main organ that absorbs ZEN since ZEN is often found in Fusarium contaminated food. This means the intestinal mucosal barrier will be the first to be affected by ZEN. Although I have not found human studies, there are some animal studies around ZEN and it's affects on the gastrointestinal environment. There is a mouse study where short-term ZEN exposure caused intestinal flora imbalance and altered intestinal mucosal immune function, inducing a severe mucosal inflammatory response, which caused microscopic changes in the small intestine.  There are wide species differences in how ZEN is processed, but there have been shown to be estrogenic affects in all animals/humans to some degree and in studes done so far there are also short term digestive issues in various animals and ZEN may be a potential factor in inducing inflammatory diseases in the human intestinal tract also.

 

How The Body Transforms And Removes The Mycotoxin Zearalenone

Zearalenone has  a complex metabolism. It is rapidly and well absorbed after oral ingestion, being metabolized at the intestinal level, in hepatic tissue and probably other tissues not yet studied.  Wide interspecies differences in zen absorption, distribution, metabolism and excretion have been documented. It has a variety of degradation and conjugation products. Uptake in a pig after a single oral dose of 10 mg/kg was estimated to be 80–85% with an extensive biliary excretion. Absorbtion of ZEN, alpha-ZAL and the glucuronides and sulfates of ZEN and alpha-ZAL hae been shown to be rapid from the intestine.

Biliary excretion with entero-hepatic circulation occurs in rats and mice, while urinary excretion predominates in rabbits.Pigs have entero-hepatic circulation of ZEN but the main route of excretion in pigs is urinary. Limited data suggests humans also have significant urinary excretion.

Zearalenone may undergo modification in plants, fungi and animals by phase I and phase II metabolism. Modified forms of ZEN found in feed include its reduced phase I metabolites (i.e., α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol) and its phase II conjugates (Phase II conjugate forms with sulfate and glucuronic acid).

Conversion of ZEN into metabolites has been shown to take place in the intestinal mucosa as well as the liver. However, the gastrointestinal flora are also involved. They appear to be deconjugating the phase II forms of glucuronides, sulfates and glucosides.   Hydrolysis and deconjugation are dependant on animal species and, diet variation affect on the gut flora. As mentioned previously, it is known that ZEN and it's metabolites are rapidly absorbed by the intestinal tract.

Phase I metabolism of hydoxylation via 3α- and 3β-hydroxysteroid dehydrogenases  create toxic reductive metabolites that may be greater than the estrogenic potency of ZEN,  Phase II biotransformation (conjugation) produces decreased toxic biological activity in the form of ZEN glucuronides and sulfates compared to ZEN and unconjugated ZEN metabolites. As with many mycotoxins, the conjugated forms are less toxic. In the case of ZEN, conjugation is with the help of uridine diphosphate-glucuronosyltransferase (enzyme used for glucuronidation) and sulfotransferase (enzyme used for sulfation) creating conjugated  forms with glucuronic acid and sulfate.

There is a question as to if ZEN goes through similar recycling of conjugation and hydrolysis forms like estrogen does. Indeed it is deconjugated by intestinal microflora.

Both free and conjugated forms of ZEN and its metabolites need to be tested for to get the whole ZEN picture.

Examining The Biotrasformation Pathways For Zearalenone Mycotoxin

  • Phase I hydroxylation catalyzed by 3α- and 3β- hydroxysteroid dehydrogenase which creates Alpha and Beta-Zearalenol
  • Phase II conjugation of zearalenol  with glucuronic acid, and less so with sulfation.

We know that Phase I creates some metabolites that are more toxic than ZEN itself. So, phase II becomes very important to dimish their toxicity through conjugation of the phase I metabolites. Phase II conjugation of ZEN and it's metabolites is largely via Sulfation and glucuronidation which are conjugation processes catalysed by sulfotransferases (SULTs) or uridine diphosphate glucuronosyltransferases (UGTs), respectively. Glucuronidation is the pathway usually used to conjugate ZEN and therefore how well a human or animal is able to glucuronidate zearalenone makes a difference as to how much it affects them. Pigs have been shown to be more sensitive to zearalenone endocrine effects due to their greater incidence of hydroxylation to alpha-zearanol and low glucronidation capacity compared to other animals. Rats and cows have decreased sensitivity to estgenic effects of ZEN partly due to their increased conversion of zearalenone to beta-zearalenol. Some humans have decreased ability to glucuronidate also. Infants often have immature abilities of glucuronidation. Gilbert's Syndrome is another situation where glucuronidation becomes an issue and although thought to be a non-issue in the past, I think it is causing problems that have been ignored simply due to our lack of knowledge around glucuronidation in the past.

We don't have adequate knowledge of the metaoblism of most mycotoxins and this is true for ZEN. The monitoring of ZEN and metabolites in the body fluids is the next step researchers are embarking on and this will give us  some useful data. We need to know the amount of substance that is actually systemically available in relation to what is actually consumed as well as the affect on organs. Additionally, we need to know more about the metabolites and the process of how they are removed from the body.

There is great difference between toxicokinetics and metabolism between sexes, and various species of animals. Some critters are going to be much better at transforming and removing it than others. I mentioned that cattle are less sensitive than pigs as cattle form Beta-ZEL as the main metabolite while pigs form alpha-ZEL as the main metabolite. It appears that humans form these equally. There are age differences in pig metabolism with the young ones excreting mainly ZEN in the urine.

It is hard to extrapolate from animal to human, when you see quite a bit of variability in how ZEN is modified by various animals after oral ingestion.

In a study of one person ingesting ZEN, about 10% of it was excreted in urine 3-10 hours after eating. Based on this one piece of data, researchers estimate that 10-20% of an oral dose of ZEN is excreted within 24 hours. However, in reality people have vastly different abilities to detox, or biotransform toxins. Plus we know little on the metabolite toxicokinetics.

Binders For Zearalenone Mycotoxin

Binders are used to prevent absorption of zearalenone from the intestinal tract. This can be zearalenone or it's metabolites from immediate ingestion or it could be from the mycotoxins going through the enterohepatic circulation and ending back up in the intestines again. Either way, binders are used to bind to the toxins and prevent them from being absorbed. They end up in the feces, rather than be reabsorbed by the intestinal tract. For an indepth look at binders check out my other articles on binders. Some of the binders more commonly used for removing mycotoxins are clay, charcoal, cholestyramine, yeast cell walls and water soluble fibers.

Some mycotoxins such as aflatoxin B1 have a rather planar structure, which facilitates interlayer adsorption, whereas ZEN has a more spherical geometric structure and makes a clay such as montmorillonite clay a poor adsorbing medium for ZEN even though it is great for aflatoxin B1. Additionally aflatoxin B1 is more hydrophilic than ZEN and the clays interlayer space is hydrophilic, again making the clay a better binder for the aflatoxin than ZEN. For these reasons clays and other similar materials have not shown good binding capabilities for ZEN.

A Good Binder Has These Characteristics
  • Adsorption capacity
  • Specificity
  • Irreversible binding
  • Safety
  • In vivo studies support use for the toxin in question

Adsorption capacity - The ability of the binder to bind to the mycotoxin in question.

Specificity - How specific the binder is for the target mycotoxin and not other things such as nutrients.

Irreversible binding - If a binder easilly becomes unbound, it can be released again.

Safety - Obviously, we don't want to use toxic binders.

In Vivo Studies - I need to explain that in vivo means in the body studies, rather than in vitro which is in a petri dish. Some binders work in a petri dish but not in the body. In vitro is easy to research and is a good starting place and if it is all we have, well we might try it, but in vivo research is always best. Often binders work in vitro, but not in vivo. Results can also change depending on the individual characetristics of the animal/human too.

Clays Not So Good In Most Studies

You will find a few studies that look promising. For instance, there is a chicken study showing efficacy of  using montmorillonite clay adsorbent Calibrin-A (CA)  with chickens given ZEN in their diet that looked like it might be useful.  However, another montmorillonite clay study in mice given a zearlenone diet actually showed increase uptake of ZEN and increased toxicity from ZEN with the clay. This was one of the modified clays that are often sold. Most of the studies are on modified clays, rather than natural clays. It was a low-pH montmorillonite clay exchanged with alkylammonium compounds. Unfortunately, many of the clays used in the research are modified clays companies are studying in hopes to have something that works great that they can patent. This makes the studies more confusing.  Also, labeling can be deceiving on the clays for sale. You may not always know what you are getting unless you are careful about sourcing as they are often mislabeled as natural since natural means absolutely nothing legally.

Cholestyramine- Maybe

Cholestyramine was proven to be an effective binder for zearalenone in vitro, which was confirmed in experiments using a dynamic gastrointestinal model. This is not an in vivo experiment but does get closer to real life than usual in vitro testing.

Zeolites - Confusing, but appears not so good over all

Zeolites have both been shown to be useful in adsorption of ZEN as well as useless. The treated zeolites seem to have some of the better results. The studies are confusing and I am not really sure what to think about them.

Saccharomyces cerevisiae - Promising

Saccharomyces cerevisiae cell walls are used to bind several mycotoxins in animals foods. This includes aflotoxins, ochratoxin, and zearalenone. This reduces the absorption of the mycotoxins through the intestinal wall. It is also thought that the yeast based products may act as a probioic contributing to the general health of the animal through enhancement of the gut wall integrity and support of the immune system. The alpha mannan oligosaccharides in the S. cerevisiae cell walls have been found to be important in the role of binding the mycotoxins. Beta-D-glucan in the cell wall is also thought to possbily play a part in binding the toxins. The capacity to bind has been found to change in different strains of S. cerevisiae and the pH of the digestive tract also affects binding performance. More acidic conditions favor mycotoxin adsorption on yeast cell walls in simulated gastrointestinal environments. One study found that the more Beta-D-Glucan that was in the S. cerevisiae cell wall, the better it bound the ZEN. The chemical interactions betweenb-D-glucans and zearalenone were found to be more of an adsorption type reaction than a molecular binding reaction.

Enhancing Removal Through Increasing Biotransformation And Modifying The Zearalenone Mycotoxin

Mycotoxin modifyers  aim to alter the chemical structure of the mycotoxin rather than binding it. The idea is that altering the structure will decrease it's toxicity. Indeed if modified correctly, you can usually decrease the toxicity. Some common modifiers of mycotoxins used are enzymes, bacteria, yeasts, and supplements/herbs that enhance conjugation known to make the mycotoxin more water soluble and easier to remove from the body.

Saccharomyces cerevisiae

There is also the metabolic affect of live S. cerevisiae on zearalenone, changing into it's metaoblites. Most strains of live S. cerevisiae tested will effectively remove most of the ZEN when introduced to yeast broths. They metabolize it into other compounds. The most active strains (S. cerevisiae CS, LL74 and LL83) were able to eliminate more than 90% of ZEN present in the culture media in one day. Research found that wild strains from siliage metabolized ZEN in much the same way as commercial strains, intimating that wild strains may have the same anti-mycotoxin products as commercial strains being used. There is of course the concern that the ZEN may be changed into alpha- zearalenol which is more estrogenic than the zearalenone, but it appears beta-zeranelol is the most prevelant metabolite and it has lower estrogenic activity than zearanlenone. The research shows reason to believe that there are other metabolites being created that are not known at this time.

Enchancing glucuronidation and sulfation

Enhancing glucuronidation capacity inactivates ZEN. We know that ZEN is largely conjugated through glucuronidation and sulfation in Phase II biotransformation with an emphasis on glucuronidation. By enhancing these methods of the phase II biotransformation process and supporting glutathione production to mop up reactive oxygen species (ROS) created along the way, it seems a lot can be done to assist in removal of ZEN and protection from ROS.

We mentioned previously that gut bacteria have been shown to change the conjugated ZEN back into more toxic hydroxylated ZEN. This appears to largely be unfriendly bacteria. In regard to the glucuronidated ZEN, it may be helpful to support healthy gut bacteria to decrease this alteration. Beta-glucuronidase (breaks down glucuronide conjugates) is inducible by anaerobic E.coli, Peptostreptococcus, Bacteroides and clostridia. Using probiotics and prebiotics to increase the friendly gut bacteria and keep the unfriendly ones in check will help reduce beta-glucuronidase in the gut and keep the ZEN in it's conjugated glucuronide form. Using calcium-D-glucarate as a supplement also inhibits the enzyme.

Calcium D-glucarate

Calcium D-glucarate is a suppressant of beta-glucuronidase, and for good measure it has shown reductions in breast cancer risk in animal studies.

Calcium D-glucarate inhibits the deconjugation of glucuronides by inhibiting beta-glucuronidase. This supplement is used to enhance glucuronidation indirectly through inhibition of beta-glucuronidase. Some foods are also high in calcium D-glucarate. They include all of the mustard family vegetables such as broccoli, cabbage, Brussels sprouts, as well as apples, grapefruit and oranges.

 

Oxidative Stress And Use Of Antioxidants

Oxidative damage is thought to be a factor in cell damage and death from ZEN.  Zearalenone has been shown to affect integrity of the DNA and mitochondria, decrease cell proliferation and modulate the inflammatory response. These are usually associated wtih oxidative stress. Most studies of toxicity have shown that the cytotoxicity of ZEN is determined by the production of ROS, DNA damage, and an increased formation of lipid peroxidation.

Zearalenone has been shown to cause oxidative damage in vitro and in vivo.  Studies show it increases oxidative stress in farm animals.  There is increase reactive oxygen species (ROS), repressed activity and expression of anti-oxidative enzymes in vitro. In weaned piglets you see increased serum alanine aminotransferase, aspartate aminotransferase, γ-glutamate transferase and alkaline phosphatase concentrations in liver and serum in a ZEN dose dependant manner while at the same time the glutathione peroxidase and superoxidase dismutase and spleen weight were decreased with ZEN dietary increase. In rats ZEN exposure not only increased lipid peroxidation, but also induced oxidative DNA damage and inhibited DNA and protein syntheses.

ROS  has been implicated in the decrease in mitochondrial membrane potential and increase in DNA damage when exposed to ZEN. Using antioxidants to prevent creation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been observed to be helpful against toxic effects from several mycotoxins. Some of these tested that have shown some benefit with ZEN are  vitamin A, E, C and crocin found in saffron and Gardenia jasminoides, as well as  curcumin found in Turmeric, silymarin from Milk thistle, and lycopene have been shown to modulate oxidative stress from mycotoxins. N-aceytyl cysteine and resveratrol have also shown benefit.

Natural protective mechanisms against excessive ROS presence include activation of enzymatic (SOD, CAT, POX) and non-enzymatic antioxidants.

Specific Use Of Antioxidants

NAC

ZEN has been shown to cause increased ROS and loss of mitochondrial membrane potential and induction of apoptosis. The addition of NAC pretreatment, increased the mitochondrial membrane potential and inhibited apoptosis. Although, the research has been in vitro with NAC and zearalenone, it is common to use NAC as part of the treatment to increase glutathione for protection from oxidative stress due to other types of mycotoxins and to use it for removal of some mycotoxins by increasing glutathione for use in glutathione conjugation.

Selenium

Selenium has been shown to prevent ZEN damage to chicken spleen lymphocytes.

Treatment with selenium blocked ROS generation, improved antioxdative capacity, and reversed apoptosis and estrogen receptor stress-related genes and protein expression.

Silymarin

Silymarin, a group of constituents from the herb Milk thisle - Silybum marinanum has been shown to be protective in the face of ZEN-induced  liver toxicity and reproductive toxicity in a rat study. It was thought it might be due to the herbs improvement in the antioxidant capacity and regulation in the genes related to protein synthesis, ZEN metabolism, hormone synthesis, and ABC transporters in the tissues. The doses used were 100, 200, and 500 mg/kg and all doses showed protection.

It is my guess that many antioxidants will be found to be protective from cell and organ damage due to ZEN, but we simply have so little research currently.

Melatonin

Melatonin has shown the ability to counteract oxidative stress from ZEN.

Chrysin

Chrysin is a flavonoid found in many plants. It was given to mice at the same time they were given ZEN Chrysin attenuated the toxic effects caused by ZEN in blood and testes of mice. Chrysin treatment increased the number and motility of sperm, testosterone levels, restored antioxidant defenses and reduced the inflammation and apoptosis process.

A few places you will find chrysin is in honey, propolis and the herb Passion flower.

Kefir

The fermented milk product called Kefir showed a preventive effect of kefir against ZEN toxicity in cultured HCT-116 colorectal carcinoma cells. The co-treatment as well as the pre-treatment by kefir showed a reduction of ZEN induced damages for all tested markers. However, the pre-treatment seemed to be the most efficient, as it prevented almost all ZEN hazards associated with oxidative stress.

Testing For Zearalenone Mycotoxin Exposure

Good Testing Methods Still Being Devised

We know so little about how ZEN and it's metabolites act in the body. This makes it hard to even know how to test them. Blood levels most closely reflect internal exposure at target tissues, but blood levels of ZEN, and metabolites are relatively low, requiring sensitive analytical techniques. The enterohepatic cycling of ZEN and metabolites leads to increased levels of ZEN and its metabolites in the bile, making this the best medium to use for analytic purposes, but collecting bile on a routine basis is difficult. Urine is easy to collect, but studies in animals show that the relationship between urinary residue concentrations and dietary exposure is more variable than that of bile.

To specifically test for zearalenone exposure there are labs that offer various tests of body fluids. Urine is the most often used. You can find a variety of labs here that offer testing for various mycotoxins including zearalenone.

Where To Find Labs

There are laboratories that will look for mycotoxins in various fluids, but urine has become the most popular as it is the easiest to provide. It tells you what is being removed, but does not tell you what may still be in the body or what is cycling through the enterohepatic circulation. Still, finding zearalenone metabolites in the urine is useful data.  Many of the labs currently testing for mycotoxins include ZEN on their list.

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