Carcinogen Role of Food by Mycotoxins and Knowledge Gap
In today's world health and safety are among the basic human needs. Ensuring food safety
has been a major focus of international and national action over the last decades.
Both, microbiological and chemical risks are of concern. The "World Health Organization" (WHO) has identified as significant sources of food-borne diseases contamination of food and feed by mycotoxins (toxic metabolites of molds) and the contamination of fishery products by phycotoxins (toxins produced by algae).
Despite public health and prevention managers have paid particular attention to mycotoxins, in several areas of the world they are still an important food safety issue (Fig 1).
Fig. 1. Notifications for food and feed in 2005 (from EU rapid alert system, by European
Mycotoxins can cause diseases in humans, crops and animals that have led many Countries to establish limits on mycotoxins in food and feed to safeguard people's health, as well as the economical interests of producers and traders.
The first limits for mycotoxins were set in the late 1960s for the aflatoxins. Already approximately 100 Countries in the world have developed specific limits for mycotoxins in foodstuffs and feedstuffs and their number continues to grow (Van Egmond & Jonker, 2004; WHO, 2002a).
1.1 General information on mycotoxins
The natural fungal flora associated with foods is dominated by four genera: Aspergillus, Fusarium, Penicillium and Claviceps.
The chemical structures of mycotoxins produced by these fungi are very diverse, as are the mycotoxicoses they can cause.
The term mycotoxin was coined in 1962 after an unexplained die-off of about 100,000 turkeys.
It was then discovered that the mysterious turkey's disease was tied to a feed essentially composed of peanuts contaminated by secondary metabolites of Aspergillus flavus or aflatoxins (Bennett & Klich, 2003).
Mycotoxins are invisible, odourless molecules, and cannot be detected by taste (Binder, 2007).
It is difficult to define mycotoxin in a few words. It is a natural low-molecular-weight molecule; it is a secondary metabolite produced by molds that has adverse effects even at low concentrations on the health of humans, animals, and crops. Those metabolites constitute a toxigenically and chemically heterogeneous class.
Mycotoxins are classified from the a chemical viewpoint into: cyclopeptides, polycetoacids, terpenes, and nitrogenous metabolites, depending on their biological origin and structure (Bhat et al., 2010).
They are mainly produced by the filamentous structure of molds mycelia. They have no biochemical significance for the growth and development of the fungus itself. Over 400 mycotoxins have been isolated and chemically characterized, though research has focused on those forms causing significant harm to humans, animals and crops (Hussein & Brasel, 2001; Zain, 2011). Aflatoxins (AFLs), Ochratoxins A (OTA), trichothecenes as Vomitoxin (DON), Zearelenone (ZEA), Fumonisins B1 and B2 (FUMO B1, FUMO B2), tremorgenic toxins, and ergot alkaloids are the most important mycotoxins if we consider their effects on human health.
It is very important to note that some molds are capable of producing more than one mycotoxin and some mycotoxins are produced by more than one species of mold.
The presence of mycotoxins in cereals, wine, beef, pork and oil seed and other food products has been accepted as natural in many of the EU countries, in the U.S.A, in Canada, Russia, and in most of the Asian Countries (Bhat et al., 2010; Van Egmond & Jonker, 2004).
Mycotoxins are not destroyed by cooking and by normal industrial food processing since they all are heat-stable.
Exposure to mycotoxins may result in a variety of illnesses that fall under the heading of mycotoxicosis, from ingestion or in occupational circumstances from dermal and inhalation exposure (Jarvis & Miller, 2005; Li et al., 2011; Straus, 2011).
Human exposure to mycotoxins may occur at all levels of the food chain: via consumption of plant-derived products contaminated with toxins (cereal grains, coffee, oil seeds, spices, fruit juices, and beverages as wine and beer), or even from carrying-over of mycotoxins and their metabolites (e.g. aflatoxin M1) in milk, meat, and other products of animal origin contaminated because of using feeds with mycotoxins (Bhat et al., 2010; CAST, 2003), or by exposure to air and dust containing toxins (Jarvis, 2002;). In recent times, in actual fact, concerns have been raised about exposures to mycotoxins in indoor environments as a damp houses and buildings (Straus, 2011).
In Ferrante and colleagues study (2007) was carried out a monitoring of wheat samples because it is the main ingredient of bread and pasta, basic aliments, and quantitatively substantial of Mediterranean diet. Wheat samples, from various areas of East Sicily, was analyzed for carcinogenic micotoxins (AFLs, OTA, ZEA). The results show that all samples collected do not contained AFLs whereas OTA and ZEA have been found in the samples to medium concentrations of 0.01 mg/kg and 0.108 mg/kg, respectively. Instead all samples contained OTA and ZEA. So this study confirmed the previous findings of the related studies present in literature.
The main mycotoxins that contaminate food rarely are found in indoor environments.
Indeed, Penicillium and Aspergillus are the main contaminants of both food and damp buildings, though in the latter they produce different mycotoxins.
The genera found in outdoor air, as Aspergillus and Penicillium, are not associated with
human or animal toxicosis.
The molds associated with buildings comprise a narrow group of species that grow thanks to the nutrients present in building materials and to the available water (Jarvis & Miller, 2005; Straus, 2011).
A. versicolor, that is often encountered in damp buildings, typically produces Sterigmatocystin, a class 2B carcinogen (IARC, 1993; Jarvis & Miller, 2005).
The most frequent toxigenic fungi in Europe are Aspergillus, Penicillium and Fusarium (Bhat et al., 2010; Creppy, 2002), while the prevalence of specific toxins around the world is as follows: AFLs in Africa and in the Asian continent; AFLs and FUMO in Australia; AFLs, OTA, ZEA and DON in North America; AFLs , FUMO, OTA, DON and T-2 toxin in South America; ZEA and DON in the Eastern European Countries; OTA, ZEA and DON in the Western European regions (Bhat et al., 2010; Van Egmond & Jonker, 2004; WHO, 2002a). However, with the open global trade these mycotoxins might also be detected in all areas of the world.
Fig. 3. Distibution of mycotoxins around the world. (AFLAs: Aflatoxins; FB: Fumonisins; OTA: Ochratoxin A; ZEA: Zearalenone; T-2: T-2 toxin).
At this stage, in Europe mycotoxins are controlled, but the regulatory policies still need to be standardized in the various European Countries.
Factors contributing to the presence or production of mycotoxins in foods or feeds include storage, environmental and ecological conditions, but also farming activities, such as drying, handling, packaging and transport, contribute to molds growth and to increase the production of mycotoxins when good practices are not followed (Zain, 2011).
Researchers have found a variety of factors operating interdependently in the production of mycotoxins. Those factors have been categorized as physical, chemical, and biological.
Physical factors include environmental characteristics as temperature, relative humidity, and insect infestation.
Chemical factors include the use of fungicides or fertilizers; biological factors, instead, depend on the interactions between the colonizing toxigenic fungi and the substrate, in fact some plant species are more susceptible to colonization while environmental conditions may increase the vulnerability of others that are more resistant (Zain, 2011).
mycotoxins (including masked mycotoxins) might be monitored (Berthiller et al., 2009; Bouslimi et al., 2008; Speijers & Speijers, 2004; Tammer et al., 2007).
5. Mycotoxins control strategies, a possible form of prevention
Good agricultural practices (GAPs), good manufacturing practices (GMPs), HACCP (Hazard Analytical Critical Control Point), biological control measures and transgenic approaches, are actually the only realistic and possible form of primary prevention for mycotoxins flowering.
A control program for mycotoxins from the field to the table should include the HACCP approach, thus requiring an understanding of the interactions of the toxigenic fungi with crop plants, of the production and harvest methods for crops, of the production of livestock using grains and processed feeds, a thorough knowledge including diagnostic capabilities for mycotoxicoses, the development of new practices of foods processing for human consumption including storage and delivery (Jard et al., 2011).
A good protocol for mycotoxins checking is necessary to manage all control points and finally for being able to ensure to the consumer a food supply free from mycotoxins (Binder, 2007 ; Richard, 2007; Abrunhosa et al., 2010).
The results of HACCP show that the products obtained have an elevated hygienic quality, thus lowering the risk of contamination. The dried pasta, for example, is one of the foods most commonly consumed by Italians and Italy is leading the world ranking for the consumption of pasta. Also, pasta is conquering more and more important positions in the dietary habits of various peoples. The study of Ferrante and colleagues (2009) was to assess the presence of ZEA and OTA with the aim of identifying the consumer's risk.
The results of this study have showed OTA and Zea concentrations below the limit of methods in all types of dried pasta tested, also have showed that the HACCP system adopted by Italian food industries are adequate for preserve the food quality. This study moreover have showed in particular that the products for celiac consumers and the products made with whole wheat flour have an elevated hygienic quality.
The efficient storage systems can be also a good practice for reduction of contamination by mycotoxins.
One of the conservation techniques for food is the lyophilization. Ferrante and colleagues in 2006 carried out the study for verify presence of : tri-5 gene, ZEA, NIV and DON, in the primary products used for the preparation of lyophilized foodstuffs and in their final products (prontocrepes and prontocone).
The study have showed that the Tri-5 gene are always present in prontocrepes and always absent in prontocone products. With the air treatment a reduction of DON and NIV in both products has been obtained. The nitrogen treatment has had a single influence on the DON and in both final products, in which has had a reduction to 50 %. While, the NIV with the same treatment has reached the lowest percentage of reduction in both final products. The treatments affect relatively on the contamination of processed-foods that, therefore, in this case was due on the first quality of the primary products.
Populations residing in developed Countries are usually considered to be less exposed to mycotoxins than those in developing Countries; this might be attributed to various factors:
l execution and practice of modern food handling/preservation technology
l successful governmental regulation and commercial control over food quality and safety.
However, even monitoring and exercising of good agricultural and manufacturing practices (GAP and GMP) along with an effective HACCP approach might not completely avoid or eliminate mycotoxins in the food chain (Bhat, 2010; Jard et al., 2011; Zain, 2011).
5.1 Biological control
Various biocontrol strategies are possible for reducing the levels of mycotoxins in the crops, such as development of atoxigenic bio-control fungi that can out-compete their closely related, toxigenic strains in field environments.
It has been reported the existence of non-toxigenic strains of A. flavus and A. parasiticus that can reduce the post-harvest aflatoxin contamination by 95.9%.
The competitive use of biological agents such as F. verticillioides strains was observed to suppress the growth of fumonisin-producing fungi.
Control of fumonisins producing fungi by endophytic bacteria has also been reported and competitive exclusion was thought to be the mechanism involved.
It has also been reported that Pichia anomala, Pichia kluyveri and Hanseniaspora uvarum can inhibit in vitro OTA.
Furthermore, fungal strains of Trichoderma have been demonstrated to control pathogenic molds through mechanisms such as competition for nutrients and space, fungistasis, rhizosphere modification, mycoparasitism, biofertilization and the stimulation of plant-defense mechanisms (Bhat, 2010; Jard et al., 2011; Zain, 2011).
5.2 Chemical control
Appropriate use of pesticides during the production period could help in minimizing fungal infections or insect infestation of crops and consequently mycotoxins contamination; therefore, Fumonisins contamination could be reduced by application of fungicides that have been used for controlling Fusarium, such as prochloraz, propiconazole, epoxyconazole, tebuconazole, cyproconazole and azoxystrobin.
On the other hand, fungicides such as itraconazole and amphotericin B have been shown to effectively control the aflatoxin-producing Aspergillus species. However, use of fungicides is been discouraged because of economic, environment and severe food safety issues (Bhat, 2010; Jard et al., 2011; Zain, 2011).
Decontamination of food and feed from mycotoxins could be achieved through chemoprotection or enterosorption.
Chemoprotection from aflatoxins has been demonstrated with the use of a number of chemical compounds like oltipraz and chlorophylin or through dietary interventions based on broccoli sprouts and green tea that increase animal's detoxification processes or prevent the production of epoxide, that is known to cause chromosomal anomalies.
However, this intervention might not be sustainable in the long-term because it is expensive and shows some side effects.
Entersorption is based on the discovery of certain clay minerals, such as Novasil, that can selectively adsorb mycotoxins enough to prevent their absorption from the gastrointestinal tract .
There are different adsorption agents but their efficacy in preventing mycotoxicosis varies.
Calcium montorillonites have proven to be the most highly selective and effective of enterosorbents. However, with enterosorption, there is a risk that non-specific adsorption agents may prevent uptake of micronutrients from the food (Bhat, 2010; Jard et al., 2011; Zain, 2011).
5.4 Breeding for resistance
Breeding for resistance is the most promising and encouraged long-term strategy for control of mycotoxins contamination in Africa. Sources of resistance to A. flavus and Fusarium spp. have been identified, particularly F. verticillioides.
Zain (2011) states that "Prototypes of genetically engineered crops have been developed that
a. contain genes for resistance to the phytotoxic effects of certain trichothecenes, thus
helping reducing fungal virulence or
b. contain genes encoding fungal growth inhibitors for reducing fungal infection in the USA".
To devise effective strategies to control fungal infections and minimize mycotoxins production in host plants, a good knowledge of genetic variability and plants population structure is necessary (Kazan et. al., 2011; Jard et al., 2011; Zain, 2011).
6. Regulation of mycotoxins in foods and feeds
A series of studies on mycotoxins in food and feed have been carried out around the world in order to improve policy making.
Regulations relating to mycotoxins have been established in many Countries to protect consumers from the harmful effects of these compounds. In 2003 about 100 Countries (covering approximately 85% of the world's inhabitants) had already specific regulations or detailed guidelines on mycotoxins in food. Regulations concerned AFB1, AFB2, AFG1, AFG2 and AFM1, trichothecenes (Vomitoxin, diacetoxyscirpenol, T-2 toxin and HT-2 toxin), FB1, FB2, and FB3, agaric acid, ergot alkaloids, OTA, patulin, phomopsins and ZEA, leaving out sterigmatocystin.
Harmonized EU limits now exist for 40 mycotoxin–food combinations. The direct or indirect influence of European organizations and programs on the EU mycotoxins regulatory developments was significant.
The current maximum limits are more and more based on scientific evidence from authoritative agencies such as FAO/WHO, Joint Expert Committee on Food Additives of the United Nations (JECFA) and the European Food Safety Authority (EFSA) (Van Egmond & Jonker, 2004).
The FDA has action levels for aflatoxins but the European Community levels are more restrictive (Creppy, 2002; Mally & Dekant, 2009).
Efforts have continued internationally to establish better guidelines to control mycotoxins. FAO, for example, has worked with developing Countries (African and East-Asian Countries) to mitigate mycotoxins contamination in foods and feeds.
Many Countries have regulatory or guideline limits for OTA in foods but in the majority of cases OTA content limits have been established only for cereals and cereal products (see Tab. 5).
There are still some discussions in those organizations regarding the limit that should be established for OTA in some cereal-derived foods, in fact in some Countries there is no limit or it is not harmonized with the international level (see Tab. 5) (Araguás et al., 2005).
Table 4. Worlwide limits for AFB1 and sum of AFB1, B2, G1, G2 in food.
Many Countries, even if industrialized, such as USA and Canada, still today lack of legal limit for AFB1, showing how the problem is unfortunately underestimated.
Furthermore, looking at Table 5, it is also evident that still today in many Countries of the world there are no limits for OTA, ZEA and Fumonisins, and that worldwide regulations for sterigmatocystin are completely lacking.
Several Countries, such as Bahamas, Bolivia, Burkina Faso, Cameroon, Ecuador, Ethiopia, Iraq, Myanmar, Nicaragua, Pakistan, Panama, Qatar, Trinitad and Tobago, Uganda, United Arab Emirates, Yemen, Zambia and Zimbawe, until 2003, hadn't yet established any regulations or limits for any mycotoxins, that means that the carcinogenic risk to which local populations are subjected is high.
Table 5. Worldwide maximum limit of carcinogenic mycotoxins for food.
7. Conclusions and future research
Besides the demonstrated effects of mycotoxins on humans and animals, some important aspects of their toxicology and possible control mechanism are still unknown and unexplored. The occurrence of mycotoxins in the food chain is therefore an unavoidable and serious problem that the world is facing not without efforts nor difficulties.
The toxic effects of mycotoxins (on liver and kidney, hematopoietic toxicity, immune system toxicity, reproductive system toxicity, foetal toxicity and teratogenicity, and moreover carcinogenicity) are mostly known in experimental models but the extrapolation for humans is often inaccurate.
The inaccuracy of extrapolation for humans may be explained by the lack of adequate food consumption data and/or lack of knowledge about relative health risks associated with specifically proposed limits and finally by the gap of knowledge on the possibility of synergism with other mycotoxins present in the same food products.
Wide gaps still exist on the toxicological effects of feeding animals with mycotoxin-contaminated feeds.
Further research also needs to be focused on epidemiology of toxic effects, especially in humans.
Development of new genetically modified plants by the application of genetic engineering that might be resistant to fungal invasion might also prove today to be a good option for preventing growth of toxigenic fungi, though they leave open many question marks in any case.
Development of methods for simple, rapid and economic analysis chemical contaminants throughout the food chain. The researchers Ferrante and Oliveri Conti have carried out the development of a method for simultaneous determination of 13 mycotoxins and some their metabolites in LC-MS tandem TQD in serum and urine of humans (Article awaiting acceptance by journal) after enzyme treatment of samples.
Even though research papers, reviews, monographs, and government reports are available on the contamination of food and feed by fungal toxins, nonetheless most of the information are restricted to only one type of mycotoxin (Bhat et al., 2010), and they do not take into account ghost mycotoxins.
Much still needs to be done, both at governmental and scientific level, in order to reach higher standards of protection for consumers. It is important to harmonize as soon as possible the maximum limits for food taken from Country to Country due to global trade, that increases the risks due to mycotoxins ingestion in the population of the importing Country.
The authors gratefully acknowledge the contribution of Dr. Pasquale Di Mattia in terms of comments, medical English and constructive suggestions provided for improving the manuscript.
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Article made possible through the contribution of M. Ferrante et al., University of Catania, Department "G.F. Ingrassia" Hygiene and Public Health, Italy