Effects of Zearalenone (ZEA) and Mycotoxin Interactions on Animal and Human Health and Prevalence in the Food Supply

Abstract

Zearalenone (ZEA) is a type of estrogenic mycotoxin produced by Fusarium species. ZEA mimics the hormone, estradiol, and can significantly impact health, specifically in reproductive performance. Pigs are known to be the most heavily affected livestock species, and effects in humans have not been heavily studied. ZEA has a very common prevalence in grains across the world; however, much of the prevalence occurs in doses lower than the recommended daily intake limits set by various governmental agencies. While much of the animal feed consumed has ZEA present, it does not appear that ZEA transfers in meat or animal products, such as dairy, in high enough quantities to be over daily intake limits recognized as safe for human consumption. Nonetheless, grain for human consumption could be a concern in certain areas. ZEA is also found concurrently with another mycotoxin, Deoxynivalenol (DON), and their combined effects have also been cause for concern. Future studies should be done to ensure that limits set for daily intakes in both humans and animal species are safe, especially when considering the combined effects of ZEA and other mycotoxins. More work should be done to decrease the prevalence Fusarium mycotoxins in food items, specifically grains for both human and animal consumption. Lastly, further models and research could help producers predict when levels of ZEA would be higher based on the weather and region. 

1. Introduction

The contamination of Fusarium mycotoxins in the food supply is a very serious food safety and economic concern. Zearalenone (ZEA) is an estrogenic mycotoxin that can be commonly found in livestock grains and in the human food supply(Hueza et al., 2014 and Liu et al., 2018). ZEA is produced by several Fusarium species, including F. culmorum, F. roseum, F. cerealis, F. equiseti, F. semitectum, and F. graminearum(Hueza et al., 2014, Dänicke and Winkler, 2015). The chemical structure is known as 6-(10-hydroxy-6-oxo-trans-1-undecenyl)--resorcyclic acid lactone (C₁₈H₂₂O₅) (Dänicke and Winkler, 2015).

It is also a unique mycotoxin class as it is the only known class of mycoestrogens. It mimics the hormone, estradiol, and can disrupt reproductive cycles in various species (Gajęcka et al., 2018). ZEA has even been known to cause hyperoestrogenic syndromes in humans (Ji et al., 2018).

Studies about the effects of ZEA have been conducted in a variety of species; however, pigs are largely regarded as the best models for human diseases as their digestive systems are quite similar in comparison with other livestock species (Gajęcka et al., 2018). Furthermore, ZEA can be broken down in the rumen which has the capacity to degrade ZEA into its metabolites (Kalač, 2011). Rumen protozoa reduce ZEA to α-zearalenol, β–zearalenol, α-zearalanol, and β–zearalanol (Kiessling et al., 1984); however, α-zearalenol is thought to have 2-4 times the estrogenic effect of ZEA (Becker-Algeri et al., 2016, Meucci et al., 2011).

The aim in this review is to not only combine the effects of ZEA in animals and humans, but to also discuss the prevalence of ZEA around the world in food products, both for human and animal consumption. This review also discusses the combined effects of ZEA and other mycotoxins.

2. Effects in Animals

Zearalenone has an impact on many different mammalian species. The largest effect seen is related to reproductive cycles. Swine tend to be the species affected by ZEA toxicosis the most (Biehl et al., 1993, Hueza et al., 2014).

The mycotoxin and its metabolites do this by mimicking estradiol, a predominant form of estrogen (Gajęcka et al., 2018). In male reproduction, estradiol is essential in maintaining libido (Schulster et al., 2016). In female reproduction, estradiol regulates estrous (Flowers et al. 1987). It has been shown to have a strong uterotrophic effect in most species of mammalian origin (Hueza et al., 2014).

In pigs, the prepuberal gilts, sows in estrus, and young boars are most affected by the mycotoxin. Clinical effects are hyperestrogenism in young gilts, pseudopregnancy, delayed estrus cycling, early embryonic death, and reduced libido (Kanora and Maes, 2010, Creppy, 2002, Chang et al., 1979). In gilts, ZEA’s effects included extending the periods between estrous by 30 to 70 days (Flowers et al., 1987).

These effects can be very costly for the hog industry as farmers can only make money when they are producing new animals. Over 97% of sows and gilts in the United States are bred via artificial insemination (Bush, 2015). Farmers can accurately predict the dates of a sow or gilt’s estrus cycle and know exactly when the best timer to breed her. The implications of the mycotoxin’s effects are as follows: delayed estrus cycling will impact the farmer’s reproductive schedule. Unlike humans, pigs have a defined estrus cycle. On average, the estrus cycle occurs about every 21 days (Althouse, 2018). When a pig does not adhere to this, it can be difficult for the farmer to determine when she could get pregnant. Along the same lines, reduced libido in males can impact the about of semen that is able to be collected from a boar. Furthermore, boars are often walked through pens of sows and gilts near their estrus cycles to determine which animals are ready for breeding. This may impact the farmer’s ability to know when females are ready to breed if the boar is uninterested in the females. False pregnancies make a farmer believe a female is pregnant, so he or she will discontinue trying to breed her. This wastes time as the female will not actually be bred. Abortions and miscarriages waste time and can have serious health implications for the mother.

Besides impacting reproductive health, ZEA has been known to affect digestive health as well. A study conducted by Biehl et al. (1993) found that ZEA metabolites are excreted in bile and then reabsorbed and metabolized via swine intestinal mucosal cells. The metabolites are then further returned to the systemic system and recirculated throughout the body. This extends the effects of ZEA throughout the body. ZEA is eliminated from the body by urine and feces (Dänicke and Winkler, 2015).

In a study conducted by Liu et al. in 2018, ZEA decreased the villous height and increased the crypt depth along the jejunum in pregnant sows. This change can allow for a higher permeability of the intestinal epithelial tract, which, in turn, can increase the inflammatory response and cause further digestive harm. The study found that not only did ZEA-treated sows have decreased digestive capabilities, but the digestive tracts of the piglet fetuses also had shortened villi within their digestive tracts, indicating that they may have a reduced ability to absorb colostral nutrients. As compared to control groups, piglets also had a decreased feed intake (Liu et al., 2018).  Subsequently, when ZEA is given to pigs in high doses, the activity of goblet cells increase (Obremski et al., 2005).

Furthermore, rats can also be affected. In one study, adult female rats without ovaries were given various treatment levels of ZEA. The study found that ZEA caused alterations of the thymus and spleen lymphocyte phenotypes and even decreased peroxide production by peritoneal macrophages, like estrogen. It also caused a decrease in rat body weight that was not merely related to a decrease in food consumption (Hueza et al., 2014).

3. Effects in Humans

Not much is known about the specific effects of ZEA in humans outside of cell models and veterinary studies. There have been a few reports that indicate the presence of ZEA in food consumed by humans or in blood tests. In Puerto Rico and Italy, blood tests revealed amounts of ZEA in the blood of children that had an early onset of puberty. The children were thought to have obtained these high mycotoxin levels by eating contaminated food (Sáenz de Rodriguez et al., 1999, Massart et al., 2008). While the presence of ZEA in the blood does not necessarily indicate that the toxin caused precocious puberty, it is a similar effect to what we have seen in animal models. Furthermore, 2 separate intoxications occurred in China after the affected people consumed corn or wheat. The first occurred between 1961-1985 with 7,818 people affected. People developed a condition called “Scabby grain toxicosis” which is characterized by nausea, vomiting, abdominal pain, diarrhea, dizziness, and headache. The second was between 1984-1985. Similar symptoms to the “scabby grain toxicosis” occurred in 463 out of 600 exposed persons within 5-30 minutes after consuming the infected grain. Zearalenone and Deoxynivalenol, which will be discussed later, were found to be the cause (Peraica et al., 1999).

Several studies have also been done in human cell models. In a study by Gao et al., researchers found that ZEA caused concentration dependent DNA strand breaks in embryonic kidney cells (2013). Their research further found that the cell’s lysosome may be a primary target for this occurrence. In another study, ZEA induced a caspase-mediated mitochondria-dependent apoptotic processes in human hepatoma HepG2 cells (Bouaziz et al., 2008). Another study determined that ZEN can induce cell death through necrosis in the RAW264.7 macrophage cells used (Yu et al., 2011).

The prevalence of ZEA has also been noted. In Sweden, 252 urine samples were taken from adult participants. 249 samples, or 99%, were positive for mycotoxins or their metabolites. 69% were positive for more than one mycotoxin, and ZEA was the third-most found mycotoxin. 37% were positive for ZEA, while 21% were positive for its metabolite α-zearalenol, and 18% were positive for its metabolite -zearalenol (Wallin et al., 2015). A similar study at the Institute of Sciences in Food Production in Italy also tested urine samples for ZEA, α-zearalenol, and -zearalenol and found positive samples in 100%, 92%, and 75% of samples, respectively (Shephard et al., 2013).

Breast cancer MCF-7 cells were also used to test the toxic effects of ZEA. It was found that the estrogenic activity of ZEA promoted the growth of the cells; therefore, ZEA prevalence may contribute to an increase in breast cancer (Yu et al., 2005, Ahamed et al., 2001).

4. Occurrence in Food Products

The prevalence of ZEA among human and animal food alike is highly variable depending on the product. When determining acceptable levels in food, the following level will be used: provisional maximum tolerable daily intake (PMTDI) established by the Joint Committee Food and Agriculture Organization of the United Nations and World Health Organization for ZEA is 0.5 g/kg body weight (Meucci et al., 2011). The daily average intake of ZEA in humans can be found in Table 1.

Table 1

Daily average intake of ZEA in humans (Minervini et al., 2005).

Age Status	Limit of ZEA per g/kg body weight
Adults	0.0008 – 0.029
Children	0.006 – 0.055

4.1 Prevalence in Grains and Animal Feed

Mycotoxins are highly prevalent in cereal and cereal grain products around the world, particularly Fusarium mycotoxins such as ZEA. With an increased global supply and demand, these mycotoxins are being traded around the world, which may indicate a cause to its increasing prevalence (Zinedine et al., 2007). The prevalence of ZEA in food materials has mainly been seen in grains, such as wheat, maize, barley, corn, and oats (Zinedine et al., 2007). Maize is the grain most commonly infected with ZEA (Pleadin et al., 2012), and even accounts for 30% of the total grain crop worldwide (Tang et al., 2017). Critical concentrations of ZEA in animal feed is estimated to be 2 mg/kg for cereals and cereal products and 3 mg/kg for corn products. The daily limits for livestock can be seen in Table 2. The limits set by the European Union (EU) Commission for grains is 2000 g/kg (Chang et al., 2017).

Table 2

Daily limits of ZEA (Dänicke and Winkler, 2015).

Animal	Limit of ZEA per g/kg body weight
Piglets and Gilts	100
Sows and Finishing pigs	250
Calves, Dairy cattle, Sheep, Goats	500

In Europe, maize is the most prominent cereal at risk with high levels of contamination and incidence (Pleadin et al., 2015, Zinedine et al., 2007). Several studies and their findings can be seen in Table 3. While ZEA is found in several of the samples, even most of the maximum amounts are still lower than guidelines set by the European Commission.

Various studies in Croatia to determine the amounts of ZEA within maize have also been conducted; however, results have been quite variable. Authors have attributed this variation due to weather conditions during which the studies were conducted (Pleadin et al., 2015). Season also seems to impact the prevalence of ZEA. A study conducted in the United Kingdom looked at the prevalence of ZEA in grain imports from Argentina and France between the 2004-2007 harvest. Both countries had their highest ZEA levels after very dry periods (Scudamore and Patel, 2009).

Studies done in other areas of the world have also been quite variable; however, this could be potentially attributed to the weather conditions listed above. In a 2012 study, Kim et al. found that the prevalence of ZEA in compound feeds was 71.33% and 47% in feed ingredients. 98% of cattle compound feeds were found to be contaminated with ZEA, with the highest contamination level at 0.405 mg/kg (Kim et al., 2014). In Turkey, 180 feed samples were taken from dairy, beef, lamb-calve rations. 31.7% of those rations were positive for ZEA, with all samples being less than the 500 g/kg^-1 limitset by the Turkish government (Kocasari et al., 2012). 99% of compound feeds in South Africa were contaminated with ZEA (Burger et al., 2013).

Table 3

Prevalence of ZEA in grains around the world.

Country	Grain tested	Incidence of positives	Maximum amount of ZEA found per sample (g/kg)	Average amount of ZEA found per sample (g/kg)	Reference
Croatia	Maize	3.31%a		411	Pleadin et al., 2015
	Wheat	5.88% a		275	Pleadin et al., 2015
	Barley	0% a		1.78	Pleadin et al., 2015
	Silage	0% a		102	Pleadin et al., 2015
	Piglet Diets	0% a		21.5	Pleadin et al., 2015
	Finishing Diets	7.89% a		117	Pleadin et al., 2015
Germany	Maize	75%	1,100		Döll and Dänicke, 2011
	Cereal Grains	24%	500		Döll and Dänicke, 2011
	Piglet Diets	38%	100		Döll and Dänicke, 2011
	Sow Diets	35%	100		Döll and Dänicke, 2011
	Finishing Diets	33%	400		Döll and Dänicke, 2011
European Member States	Wheat	30%	152		Döll and Dänicke, 2011
	Maize	79%	6,492		Döll and Dänicke, 2011
	Barley	5%	53		Döll and Dänicke, 2011
	Oats	20%	1,310		Döll and Dänicke, 2011
	Rye	5%	24		Döll and Dänicke, 2011
Netherlands	Compound Feed	28%	363	80	Driehuis et al., 2008
	Silage	17%	273	125	Driehuis et al., 2008
	Feed Commodities	38%	108	80	Driehuis et al., 2008
	Forage Products	8%	82	82	Driehuis et al., 2008
Slovakia	Chicken Feed	88%		21	Labuda et al., 2005
South Korea	Grains	77%	19	277	Chang et al., 2017
	Grain By-Products	83%	288	1,072	Chang et al., 2017
	Corn Meal	82%	95	1,330	Chang et al., 2017
	Fibrous Feed	50%	285	1,315	Chang et al., 2017
	Food By-Products	62%	18	176	Chang et al., 2017
	Beans	100%	15	15	Chang et al., 2017
Kuwait	Wheat Bran	92.9%	50	46.4	Beg, et al., 2006
	Soybean Meal	100%	69.9	52.4	Beg, et al., 2006
	Yellow Maize	100%	99.6	54.2	Beg, et al., 2006
	Layer Mash	90%	84.7	48.6	Beg, et al., 2006
	Broiler Starter	92.9%	80.1	50.6	Beg, et al., 2006
	Broiler Finisher	84%	400	67.6	Beg, et al., 2006
Japan	Wheat	5.3%	151	20.5	Yoshinari et al., 2014
	What Flour	18%	3.3	1.2	Yoshinari et al., 2014
	Barley	9.8%	27.1	9	Yoshinari et al., 2014
	Job’s Tears Products	65%	153	17.5	Yoshinari et al., 2014
	Beer	0%			Yoshinari et al., 2014
	Corn Grits	70%	32.2	7.9	Yoshinari et al., 2014
	Azuki Beans	72.5%	125	44.6	Yoshinari et al., 2014
	Soybeans	0%			Yoshinari et al., 2014
	Rice with Mixed Grains	83.3%	39.3	4.3	Yoshinari et al., 2014
	Sesame Seeds	66.7%	21.3	4.5	Yoshinari et al., 2014
	Non-Dried Maize Based-Food	100%	28.5	2.35
South Africa	Dried Maize Based-Food	100%	239	11.5	Shephard et al., 2013
Brazil	Rice Grain	60%b	126.31	113.8	Savi et al., 2018

^a This incidence was only given with the incidence of positive samples higher than recommended by the European Union limits

^b This incidence is the amount of positive samples with greater than the maximum Brazilian limit of ZEA in rice (100 g/kg) (Savi et al., 2018).

4.2 Prevalence in Meat

The Food and Drug Administration (FDA) has established the concentration levels for α-zearalanol in uncooked beef to be 150 g/kg in muscle tissue, 300 g/kg in liver, 450 g/kg in kidneys, and 600 g/kg in fat (Meucci et al., 2011). In 2008, the European Food Safety Authority conducted a study on the prevalence of ZEA in their 27 Member States. 69 non-compliant results from bovine sources were identified, and feed contaminated was suspected as the origin for all non-compliant results (European Food Safety Authority, 2010).

As discussed previously, it is believed that the main route of ZEA excretion is via urination and defecation. Because of this, the significance of the prevalence of ZEA and its metabolites in meat products is thought to be very low (Meucci et al., 2011, Creppy, 2002). In the study by Pleadin et al., urine and meat samples were taken from pigs naturally exposed to ZEA (2015). The average concentration in urine was 206  20.6 g/L which is in line with the main route of ZEA excretion. The average concentration in meat was 0.62  0.14 g/kg. As compared to the TDI of 0.25 g/kg body weight per day, the effects of this are not significant in humans (Pleadin et al., 2015). A study by Vulić et al. found similar results (2012).

Another study looked at the prevalence of ZEA in chicken meat and egg samples taken from the central areas of Pubjab, Pakistan. Researchers found that 52% of chicken meat and 32% of egg samples were positive for containing ZEA. The average level of ZEN was found in chicken meat was 2.01  0.90 g/kg, and in eggs the average was 1.58  0.93 g/kg (Iqbal et al., 2014).

An Italian study looked at the prevalence of ZEA in milk-based formulas and meat-based infant food. Among the 44 meat-based foods, 27% of meat samples were detected to contain α-zearalenol, but only one sample was contaminated to a level over the standard allowed (Meucci et al., 2011).

In one study, 6740 samples of various food products with animal origins were analyzed for ZEA contamination. 22 of those products had values higher than 0.25 g/kg body weight per day – a rate of 0.33%; therefore, ZEA in food products of animal origins does not pose a significant health risk to human consumers (Dänicke and Winkler, 2015).

4.3 Prevalence in Dairy Products

The presence of ZEA in cow’s milk and cow milk products was very low. Carry-over rates from dosage to milk are very low; therefore, milk products are thought to carry very little risk to humans (Coffey et al., 2009). The low carry-over rate is thought to occur as the result of the rumen being able to break down ZEA into its metabolites (Kalač, 2011).

In one study, cows were fed specific amounts, 50mg and later 165mg, of ZEA for 21 days with a 21 day wash out period in between. Each sample proved to be negative. Researchers then increased the dose to 544.5 mg daily in which they only found a maximum concentration of 2.8 ng of conjugated ZEN/mL of plasma on day 3, which quickly declined to non-detectable levels by day 5. A dose of 544.5 mg would be too high to be in a normal cow ration; therefore, the risk to humans is also negligible (Prelusky et al. 1990).

An Argentinian study by Signorini et al. (2012) found that the concentration of ZEA in cow’s milk produced in Argentina was 0.125 ppb. Feed samples were also tested. ZEA was found in 78.8% of feed samples, and 8.9% were over the 500 ppb maximum level set by European regulations The concentration of ZEA in the tested cow’s diets was estimated at 194.9 ppb. Furthermore, in autumn, it is estimated that 13% of ZEA consumed comes from pastures, while in the spring, this percentage jumps to 56% (Signorini et al., 2012).

In another study, 185 cow’s milk-based infant formulas were analyzed for the presence of ZEA. ZEA was detected in 9% of milk samples, α-zearalenol was detected in 26% of milk samples, and β –zearalenol was detected in 28% of samples (Meucci et al., 2011).

Another study was conducted in China taking raw milk, liquid milk, and milk powder samples from Chinese dairy farms and supermarkets. While most samples contained ZEA and/or its derivate, α-zearalenol, all samples were significantly lower than the aforementioned European regulation limits (Huang et al., 2014).

5. Interactions with Deoxynivalenol and Mixed Mycotoxin Studies

Deoxynivalenol (DON), also known as vomitoxin, is another Fusarium mycotoxin. It is a trichothecene and is one of the most common mycotoxins in various cereal grains, such as wheat, corn, and barley. Its chemical formula is 12, 13-epoxy-3,7,15-trihydroxy-trichothec-9-en-8-one (Maresca and Fantini, 2010). It has been known to cause anorexia, vomiting, and limited immune function in livestock species when DON-contaminated grain is eaten (Ji et al., 2018). DON is extremely stable as well and does not degrade at high temperatures (Rotter, 1996). It also can inhibit the synthesis of proteins, and important step during growth and development of animals (Gajęcka et al., 2018).  DON and ZEA are frequently produced at the same time (Rotter, 1996) and occur in similar food sources (Dänicke and Winkler, 2015, Driehui et al., 2008).  They can also have synergistic cytotoxic effects when combined (Ficheux et al., 2012), Kouadio et al., 2007).

Like Zearalenone, it also can pose a significant risk to swine health (Dänicke and Winkler, 2015). In a study conducted in mice by Ji et al., the combined effects of DON and ZEA were shown to have a potentially antagonist effect in galactose metabolism and the tricarboxylic acid cycle (TCA) (2018). DON was also shown to alter the production of mucus in patients with celiac disease or inflammatory bowel disease (IBD) when associated with ZEA (Maresca and Fantini, 2010). Furthermore, when DON and ZEA were exposed to pigs in low doses with the T-2 toxin, increased the amount of mucus production occurred, by increasing the number of mucus-producing cells (Obremski et al., 2008).

In another study, the exposure to both DON and ZEA caused serious physiological effects in pigs. DON and ZEA were fed at the published critical values cited earlier in this paper. Liver and organ damage, along with increased -glutamyltransferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) was observed. Histopathology on the subjects’ organs indicated multi-organ toxicity among the liver, spleen, lymph nodes, uterus, and kidneys (Chen et al., 2008).

The interaction of DON and ZEA can also be detrimental to human health. As discussed in section 3 of this paper, DON and ZEA were found to cause serious gastrointestinal distress among Chinese people that consumed contaminated grain. Further studies in cell models show that this combination of mycotoxins can be detrimental. One study found that chronic exposure to the maximum limits of DON and ZEA set by the European Union caused major changes to the metabolic activity of the HerpaRG human liver cell line (Smith et al., 2017). In a study by Kouadio et al., researchers found that the mixture of DON and ZEA induce DNA fragmentation in the human intestinal cell line, Caco-2 (2007). In the same study, another Fusarium mycotoxin, Fumonisin B1, was tested for its effects with DON and ZEA. The study found that the effects of all 3 mycotoxins created an even larger impact on the DNA fragmentation in the cell line studied (Kouadio et al., 2007). In a Swedish study, Fumoninsin B2 was commonly found with ZEA as well (Wallin et al., 2015).

Other mixed mycotoxin effects have been studied via mathematical modeling. In one study, the effects of ZEA and Ochratoxin A (OTA) were analyzed with nonlinear regression to determine their cytotoxicity in human hepatoma HepG2 cells and immortalized murine ovarian granular KK-1 cells. OTA is known for its impact on the liver, which is why the HepG2 cells were used, while the KK-1 cells were used for ZEA. The modeling systems showed that the combination of OTA and ZEA have an additivity property, and at different concentrations, may even have an antagonistic or synergistic effect (Li et al., 2014).

Predictive models have also been developed, specifically to track the occurrence of mycotoxins based on weather patterns (Streit et al., 2012).

6. Conclusion

The presence of Zearalenone in grains is still very high. Future work should focus on the reduction of ZEA among grains, whether for human or animal consumption. The effects of weather may provide insights on how to limit the amount of ZEA being produced. As grains are likely where the contamination in animal products are coming from, this will likely make meat products even less of a concern. Furthermore, even in studies where the daily limits for ZEA were studied, there were still significant health impacts to the animals. TDIs should potentially be reevaluated among species, including humans. There is very little research in the prevalence of ZEA in meat and animal products around the world as well. More research should be conducted to determine the long-term effects of exposure, especially in humans.

Lastly, more work should be done to determine the effects of ZEA with other mycotoxins, besides DON, such as aflatoxin or ochratoxin.

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