Alternaria Mycotoxins in Food and Feed : An Overview

Alternaria is one of the major mycotoxigenic fungal genera with more than 70 reported metabolites. Alternaria mycotoxins showednotably toxicity, such asmutagenicity, carcinogenicity, induction ofDNAstrand break, sphingolipidmetabolismdisruption, or inhibition of enzymes activity and photophosphorylation. This review reports on the toxicity, stability, metabolism, current analytical methods, and prevalence of Alternariamycotoxins in food and feed through the most recent published research. Half of the publications were focused on fruits, vegetables, and derived products—mainly tomato and apples—while cereals and cereal byproducts represented 38%. The most studied compounds were alternariol, alternariol methyl ether, tentoxin, and tenuazonic acid, but altenuene, altertoxins (I, II, and III), and macrosporin have been gaining importance in recent years. Solid-liquid extraction (50%) with acetonitrile or ethyl acetate was the most common extraction methodology, followed by QuEChERS and dilutiondirect injection (both 14%). Highand ultraperformance liquid chromatography coupled with tandem mass spectrometry was the predominant determination technique (80%). The highest levels of alternariol and alternariol methyl ether were found in lentils, oilseeds, tomatoes, carrots, juices, wines, and cereals. Tenuazonic acid highest levels were detected in cereals followed by beer, while alternariol, alternariol methyl ether, tenuazonic acid, and tentoxin were found in legumes, nuts, and oilseeds.


Generalities of Alternaria
1.1.Introduction.Alternaria is a fungal genus that includes saprophytic and pathogenic species and that is widespread in nature.They can infect a wide variety of crops in the field and in the postharvest stage causing considerable losses due to fruit and vegetable decay [1].The most common Alternaria species include A. alternata, A. tenuissima, A. arborescens, A. radicina, A. brassicae, A. brassicicola, and A. infectoria.They colonize a range of plants including cereals, tomatoes, apples, grapes, oil crops, oilseeds, sunflower seeds, oranges, lemons, melons, cucumbers, cauliflowers, peppers, and tangerines [2].
The optimum growth temperatures for Alternaria range from 22 to 30 ∘ C; however, minimum growth temperatures ranging from 2.5 to 6.5 ∘ C and, even lower, from 0 to −5 ∘ C in cooler regions were reported [3].Their ubiquitous occurrence and ability to grow and produce toxins even under unfavorable conditions (low temperatures and low water activity) make the Alternaria genus responsible for the spoilage of several commodities during transport and storage, even if they are refrigerated [1].Therefore, Alternaria species has been shown to be a significant contaminant of refrigerated fruits, vegetables, and stored feedstuffs [4]. A. alternata is the most important within the genus as regards to mycotoxin contamination of fruits and vegetables.However, the production of host specific toxins from pathogenic A. alternata strains seems not to be a real problem in terms of food safety while a much more important problem is its saprotrophic strains, which colonizes harvested plant products and can produce reasonable amounts of certain mycotoxins, which exert poisonous effects after consumption by humans [5].
Alternaria species have the ability to produce more than 70 toxins, which play important roles in fungal pathogenicity and food safety, since some of them are harmful to humans and animals [6].The studied Alternaria secondary metabolites belong to diverse chemical groups such as nitrogen-containing compounds (amide, cyclopeptides, etc.), steroids, terpenoids, pyranones, quinines, and phenolics [7].The major Alternaria toxins belong to the chemical groups dibenzo--pyrones, which include alternariol (AOH) and alternariol monomethyl ether (AME) and cyclic tetrapeptides represented by tentoxin (TEN).These mycotoxins were the most commonly studied metabolites produced by Alternaria strains on different substrates (tomato, wheat, blueberries, walnuts, etc.) and some of the main Alternaria compounds thought to pose a risk to human and animal health because of their known toxicity and their frequent presence as natural contaminants in food [8].
In the last decade, Ostry [1], Scott et al. [9], and Fernández-Cruz et al. [10] have studied the occurrence of the major Alternaria mycotoxins: AOH, AME, and TEN.However, food-relevant Alternaria species are able to produce many more metabolites including that known as emerging Alternaria mycotoxins described as potentially hazardous.
1.2.Toxicity of Alternaria Mycotoxins.Toxicological data are limited to the above-mentioned major mycotoxins, and even these data are incomplete, with neither good bioavailability studies nor long-term clinical studies available [12].Although little is known so far about their properties and toxicological mechanisms, bioavailability, and stability in the digestive tract, Alternaria toxins have been shown to have harmful effects in animals, including cytotoxicity, fetotoxicity, and teratogenicity.They have been related to a range of pathologies from hematological disorders to esophageal cancer.They are also mutagenic, clastogenic, and estrogenic in microbial and mammalian cell systems and tumorigenic in rats [1,13].
The benzopyrone group is the most studied among all the Alternaria mycotoxins, and it was the first to be analyzed.This group encompasses the two major toxins AOH and AME, as well as ALT and AS.Although their toxicity is not fully understood and varies from one cell system to another, AOH and AME toxicity have been identified in various in vitro and in vivo systems [14,15].Lehmann et al. [16] reported the AOH estrogenic potential and its inhibitory effects on cell proliferation.Furthermore, AOH induced marked phenotypic changes in mice macrophages, which could not be directly linked to an initial AOH-induced ROS production, cell cycle arrest or autophagy as seen as a consequence of AOH-induced double-stranded DNA breaks [17].AME and AOH were not very acutely toxic; however, they do exert genotoxic, mutagenic, and carcinogenic properties and show remarkable cytotoxicity in microbial and mammalian cell culture.Moreover, AOH and AME were able to induce DNA strand break and gene mutations in cultured human and animal cells [18,19].They interfered with the activity of human topoisomerases by affecting the stabilization of topoisomerase II-DNA-intermediates and the modulation of the redox balance in human colon carcinoma cells yet without any apparent negative impact on DNA integrity [20].AOH and AME are frequently found in combination [21] as they share a common biosynthesis pathway [22].Exposure of HCT116 cells to low AOH and AME concentrations resulted in loss in cell viability throughout the activation of the mitochondrial apoptotic process associated with the opening of the mitochondrial permeability transition pore and the loss of mitochondrial transmembrane potential.Higher toxic potential indicated synergetic effects when applied together [23].Thus, AOH and AME levels decreased cell viability in Caco-2 cells through being more cytotoxic the binary combination [24].AOH cytotoxicity decreased in Caco-2 cells in simultaneous combination with Soyasaponin I [25].
Although there have been no in vivo genotoxicity or carcinogenicity studies on experimental animals or humans for Alternaria toxins, some indications of precancerous changes have been reported in esophageal mice mucosa.Their presence in cereal grain has been suggested to be associated with high levels of human esophageal cancer in China, as well as in areas of Africa where high levels of Alternaria alternata contamination have been found [26].The underlying mechanisms of action have not yet been fully clarified.Limited data are available for long-term toxicity effects of low concentrations of Alternaria toxins and their synergistic effect on other contaminants [13].ALT has shown high acute toxicity with a LD 50 value of 50 mg/kg bw in mice, whereas TeA is considered to be the most acutely toxic among the Alternaria mycotoxins [27].TeA is known to occur in high concentrations in commodities and has attracted increasing attention in recent years [11].It inhibits protein biosynthesis at the ribosomal level in mammalian cells by suppressing the release of newly formed proteins from the ribosomes and is biologically active as it exerts cytotoxic, phytotoxic, antitumor, antiviral, antibiotic, and antibacterial effects [28].TeA toxicity has been reported in chick embryos and several animal species, including guinea pigs, mice, rabbits, dogs, and rhesus monkeys [29].TeA is acutely toxic in living organisms and its LD 50 value in mice is similar to that of the Fusarium mycotoxin deoxynivalenol (DON) [30].TeA showed acute toxic effects in rodents (oral LD 50 for mice, 81-186 mg/kg bw; and for rats, 168-180 mg/kg bw) and chicken embryos (LD 50 0.55 mg/egg).A short-term animal trial (33 days) on monkeys led to vomiting, bloody diarrhea, and hemorrhagic lesions in the intestinal tract after a treatment with 89.6 mg TeA/kg bw per day [31].Additionally, TeA has been made responsible for the outbreak of onyalay, a human hematologic disorder disease occurring in Africa, and esophageal cancer in Linxian Province, China [29].There are also reports of the interacting effects of binary combinations of TeA with Fusarium toxins (DON, nivalenol, zearalenone, enniatin B, and aurofusarin) in Caco-2 cells, leading to a decrease in cytotoxicity, compared to the expected synergistic effects.Especially when combined with DON, TeA was found to significantly reduce the cytotoxicity of this mycotoxin [32].
TeA epimerizes to a mixture of TeA and allo-TeA when treated with bases and under acidic conditions.The presence of allo-TeA in fungal culture extracts (Alternaria brassicicola, Alternaria raphani, and Phoma sorghina) has been shown.TeA and allo-TeA phytotoxic effects can be explained by the inhibition of photosynthesis by blocking the electron flow, although there are no further toxicological data regarding allo-TeA in the published literature [31].Different tautomers of 3-acetyl tetramic acid have been reported but it was not possible to distinguish between them.The TeA structure is still not clear, especially in an aqueous solution.Varying TeA structures are still widely used, resulting in different CAS registry numbers [33].
The perylenquinone derivatives, such as ATX I, ATX II, ATX III, Alterperylenol (ALTCH; synonym Alteichin), and stemphyltoxins (STE) are minor metabolites of Alternaria spp.but are considered to be very critical because of their mutagenic properties [34].Because of the lack of available reference compounds, in particular for ATXs, analytical studies remain less common [11].ATXs have been reported to be highly potent mutagens and more acutely toxic to mice than AOH and AME and cause DNA strand breaks.Recently, high genotoxic potency of ATX II in both mammalian and human cells was demonstrated, and it was described as the most potent substance within the ATX group, capable of different mechanisms of action [34].Nevertheless, data concerning the underlying modes of action are still limited [20].
Alternaria alternata lycopersici toxins (AALs) exhibit mostly phytotoxic effects but have been shown to disturb the sphingolipid metabolism in a similar way to fumonisins, which have been correlated with esophageal cancer and other animal diseases [35,36].
Altenusin (ALN) is a biphenyl derivative isolated from different species of fungi, which presents antioxidant properties and the ability to inhibit several enzymes, such as myosin light chain kinase, sphingomyelinase, acetylcholinesterase, HIV-1 integrase and trypanothione reductase, cFMS kinase, and pp60c-SRc kinase in the low micromolar concentration range, and it may serve as a chemotherapeutic agent to treat trypanosomiasis and leishmaniasis [37,38].The biphenyl basic skeleton of ALN containing a salicylic moiety and a catechol moiety could be the important part because of its interesting azole-synergistic activity.
Altersetin (ALS) was reported to possess broad antimicrobial activity against several multidrug-resistant bacterial demonstrating potent activity against several pathogenic Gram-positive bacteria and moderate in vivo efficacy in a murine sepsis model [39].
Anthraquinones and tetrahydroanthraquinone analytes are secondary metabolites widely distributed in natural biosources, which show significant biological activity.So far, several compounds of the alterporriol and altersolanol families have been reported, including the emerging Alternaria toxins macrosporin A and altersolanol A (As-A) [40], which exhibited antibacterial activity [41].
The cyclic tetrapeptide TEN is one of the major Alternaria toxins produced, along with dihydrotentoxin (DH-TEN) and isotentoxin (iso-TEN).Their structures differ at the unsaturated bond of the N-methyldehydrophenylalanine moiety, which is hydrogenated into a single bond in DH-TEN and E configured in iso-TEN.All three compounds are considered to be phytotoxins, with TEN being the most potent, inhibiting photophosphorylation and inducing chlorosis.However, no toxicological data are available for mammals, and the data on the occurrence of this toxin in food and feed are limited as well [11].
The altenuic acids consist of three closely related isomeric colorless substances (altenuic acids I, II, and III) containing one carbon-methyl and one methoxyl group.The structures of the altenuic acids I and III remain unknown, but their molecular formula (C 15 H 14 O 8 ), which is identical to that of altenuic acid II, has been determined [42].Williams and Thomas determined the chemical structure of altenuic acid II in 1973 by X-ray crystallographic analysis [43].
Monocerin is a polyketide metabolite isolated from several fungal species showing antifungal, phytotoxic, insecticidal, and plant pathogenic properties [44].Monocerin and its analogues were proven to be nonspecific toxins and nonspecific seed germination inhibitors by their interference with selected stages of cell division cycles.In recent years, they have attracted greater interest; consequently, several syntheses of this molecule have been reported [45].
Other Alternaria metabolites were reported to be phytotoxins, that is, being toxic to plants, while the role of many others, such as infectopyrones, phomapyrones, and novaezelandins, is still unknown.However, Alternaria spp.produce a variety of other metabolites for which no reports are available due to the lack of pure substances, of which only AME, TeA, AOH, TEN, and ALT are now available on the market [12].Figure 1 shows the chemical structure of the main Alternaria mycotoxins.

Stability of Alternaria Mycotoxins.
The stability of Alternaria toxins has not been studied in detail.Current information reveals that Alternaria mycotoxins were barely degraded during wet baking, while significant degradation occurs upon dry baking, with stability decreasing in the ratio of AME > AOH > ALT [46].Dibenzo--pyrones AOH and AME were stable to heating at 100 ∘ C in sunflower flour, while heat treatment at 121 ∘ C for 60 min significantly reduced the concentrations of these toxins in Alternaria contaminated sunflower flour.However, the heat-treated material caused some toxic effects when fed to rats [2].AOH and AME were very stable in spiked apple juice at room temperature for up to five weeks and at 80 ∘ C after 20 min.They were also stable in spiked white wine for almost 8 days at room temperature.ATX-1 contents were stable when added to apple juice for up to 27 days at room temperature [10].Overall, some Alternaria toxins remain difficult to degrade or decontaminate in fruits and their processed products.

Metabolism of Alternaria Mycotoxins.
Mycotoxins can be partially metabolized in living organisms, leading to the formation of conjugated toxins by the conjugation of the parent compound with glucose, sulfates, and other sugar moieties.The term "masked mycotoxins" firstly appeared defining a mycotoxin derivative that may be cleaved during digestion in living organisms to release its parent form.However, these conjugated mycotoxins are currently known as "modified mycotoxins" after a more recent comprehensive classification [47].It is not clear if these modified mycotoxins can be hydrolyzed and absorbed in the gastrointestinal tract, thereby further contributing to the overall exposure.Since current information on the bioavailability of modified mycotoxins is very limited, EFSA has recommended to national agencies that they gather occurrence data on these modified forms using properly validated, robust, and sensitive routine analytical methods [48], a prerequisite being the availability of reference standards for these compounds.Over the last two decades, formation of glycosides and sulfated mycotoxins has been reported and (bio)organic synthesis has already been applied to obtain reasonable amounts of selected conjugates as reference materials for further studies [49].Little information about the metabolism of AOH is available, but since all products of aromatic hydroxylation of AOH are catechols or hydroquinones, which can create reactive semiquinones and quinones or undergo redox cycle, its inactivation by methylation or glucuronidation/sulfation is considered to be a possible toxicological product that may cause toxic effects on cells lines [50].In the mammalian organism, formation of glucuronides is a major pathway of detoxification and excretion.Both AOH and AME have free hydroxyl groups available for metabolic conjugation.Consequently, modified Alternaria mycotoxins such as alternariol-3-glucoside (AOH3G), alternariol-3-sulfate (AOH3S), alternariol monomethyl ether-3-glucoside (AME3G), and alternariol monomethyl ether-3-sulfate (AME3S) have received more attention during the last decade [49].In vitro studies have shown that AOH and AME were readily converted to glucuronides upon incubation with hepatic and intestinal microsomes from humans, rats, or pigs in the presence of UDPglucuronosyltransferases.AME was predominantly converted to the 3-O-glucuronide whereas AOH gave rise to comparable amounts of 3-O-glucuronide and 9-Oglucuronide [51].The hydroxylation of AOH and AME was studied under in vivo-like conditions in precision-cut rat liver slices of rats.The pattern of in vivo metabolites was comparable to that of in vitro metabolites of AOH, clearly supporting the relevance of an oxidative in vivo metabolism [20].AOH and AME were extensively conjugated in suspension cultures of tobacco BY-2 cells, demonstrating that masked mycotoxins of AOH and AME can be formed in plant cells [51].Five AOH conjugates were isolated and identified by MS and NMR spectroscopy as -D-glucosides (attached in AOH 3-or 9position).For AME, conjugation resulted in -D-glucoside (mainly attached in the AME 3-position) [52].Knowledge of the toxicity and disposition of the oxidative AME and AOH metabolites is mandatory for a better understanding of the health risks posed by these Alternaria toxins, since toxicity properties may be enhanced or attenuated for their metabolites [53].Toxicokinetic studies have so far focused on toxins with a dibenzo--pyrone structure, in particular, AOH and AME.Only very little information is available on the occurrence and toxicology of Alternaria toxins with a perylene quinone structure, such as ATXs, ALTCH, and STEs.After studying the absorption of four Alternaria toxins with perylenequinone structures in the Caco-2 cell Transwell system (a widely accepted in vitro model for human intestinal absorption and metabolism), it was shown that ATX I and ALTCH were not metabolized in Caco-2 cells, while ATX II and STE III were partly biotransformed by reductive deepoxidation to ATX I and ALTCH metabolites, respectively [54].Very low ATX-II absorption and partial metabolism of ATX-I was observed in the intestinal Caco-2 Transwell system [20].
A comparative toxicokinetic study showed that TeA was completely bioavailable after oral administration in both pig and broiler chicken.Absorption was deemed to be slower in broiler chickens (.max 0.32 h in pigs versus 2.60 h in chickens) and TeA was more slowly eliminated in broiler chickens ( 1/2el 0.55 h in pigs versus 2.45 h after oral administration).These observations were mainly due to the significantly lower total body clearance (Cl 446.1 ml/h/kg in pigs versus 59.2 ml/h/kg in chickens after oral administration) [55].Concerning human exposure, the presence of TeA in the urine of six human volunteers in concentrations ranging from 1.3 to 17.3 g/L was reported.It was observed that 87-93% of orally consumed TeA was excreted as parent compound into the urine of two human volunteers within 24 h after ingestion [28].

Material and Methods
A systematic literature search was conducted using the databases Medline, Web of Science, and Scopus with the focus on the following keywords: Alternaria mycotoxins analysis, determination, occurrence, toxicity, stability, metabolism, etc.The period of time framed was 2005-2017.Fifty-eight articles, which met the criteria to be included into the study, were analyzed and classified.To facilitate data presentation three groups were established based on the analyzed food/feed matrices, namely, (i) cereals and cereal by-products, (ii) vegetables, fruits, and derived products (including juices, wine, vegetable seeds, spices, herbal infusions, and dry fruits), and (iii) mixed matrices studies, which combine the analysis of different foods belonging to both groups (i) and (ii), and products nonclassifiable into groups (i) and (ii).The information was double-checked to select bibliographies of relevant literature, and a thorough evaluation was performed to summarize the information about extraction method, analytical methodology, and studied mycotoxins limits of detection quantitation.

Analytical Methods for Alternaria Mycotoxins Determination.
Mycotoxin regulations are based on risk assessment (hazard and exposure), the parameters of which are still hard to establish, meaning that a concrete interpretation of the consequences for consumer's health remains elusive.Furthermore, it remains difficult to detect toxin metabolites at low levels in complex food matrices.Therefore, validated analytical methods ensuring robustness, sensitivity, and reliability are needed.
Although basic SLE/LLE represented approximately 50% of the extraction techniques for Alternaria mycotoxins analysis in food and feed, other used methodologies such as QuEChERS (14%), dilution and/or direct injection (11%), and the combination of SLE-SPE (14%) were of relevant importance.Direct SPE, microscale extraction, dispersive liquid-liquid microextraction (DLLME), and countercurrent chromatography (CCC) technique were also used (all <5%).Variations in the predominance of each methodology were observed depending on the analyzed food group.Thus, Alternaria mycotoxins were mainly extracted from cereals and derived products by SLE (64%) QuEChERS procedure (18%) and SLE-SPE (14%), while fruits and vegetables were mainly represented by SLE/LLE (43%), dilution-injection (16%), and SLE-SPE and direct SPE (both 14%).Other matrices or food combinations were mainly extracted by SLE/LLE and dilution-injection (both 29%), followed by direct SPE and SLE-SPE (both 14%).
With regard to mycotoxins determination, liquid chromatography (LC) was by far the most used technique, from other generally reported chromatographic techniques for, such as thin-layer chromatography (TLC) or gas chromatography (GC).High-and ultraperformance LC (HPLC and UPLC) have provided new possibilities allowing highthroughput screening by shortening the analysis time, while maintaining the chromatographic principles and improving the speed, sensitivity, and resolution [56].In the early years, atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and LC-tandem mass spectrometry (MS/MS) have become the methods of choice for the identification and quantification of these toxins [57].
Consequently, UPLC and HPLC, coupled to MS/MS (including high resolution MS; HRMS and time-of-flight MS; QTOF-MS), represented the 80% of the determinations, rising to 95% when only considering cereals and cereal byproducts.Other detectors such as ultraviolet (UV: 7%) and diode array detection (DAD: 7%) were also used, followed by the enzyme immunoassay technique (EIA: 4%).In the case of Alternaria mycotoxins detection in fruits and vegetables the main representative one also was MS/MS (64%), followed by UV and DAD (both 14%) and finally EIA (7%).
Among all of the analyzed matrices, tomato, followed by cereals and fruits, especially apples and berries, were the most studied foodstuff in recent years for Alternaria mycotoxins.Alternaria toxins are, together with aflatoxins, ochratoxin A, and patulin, the most commonly found mycotoxins in fruits and their processed products.Due to the limitations of current industrial processes to completely eliminate the rotten tissues and the reported stability of some Alternaria mycotoxins (i.e., AOH, AME, and ATXs) in fruit juices and during tomato processing, it is obvious that these mycotoxins are likely to be present in commercial end products [47].
The maximum concentrations of Alternaria toxins reported in commercial food products ranged from 1 up to 8700 g/kg.The highest levels were found in samples visibly infected with Alternaria rot, that is, in products obviously not suitable for human consumption.According to the European Food Safety Authority (EFSA), major contributors to Alternaria mycotoxins dietary exposure are grains and grain-based products, especially wheat.The highest levels of AOH and AME were found in lentils, oilseeds, and tomato paste, followed by fruit and vegetable juices, wines, cereals, and vegetables (tomatoes and carrots).The highest concentrations of TeA were reported for cereals, followed by commercial beers, while AOH, AME, TeA, and TEN were found in legumes, nuts, and oilseeds, particularly in sunflower seeds [6].
Moreover, it was observed that the cooccurrence of Alternaria toxins with other mycotoxins is plausible.Owing to the different physical and chemical properties of all the prevalent mycotoxins in fruits, the determination of trace amounts of all them in food represents an extremely challenging task [100].3.3.Most Reported Mycotoxins.Among the 58 studies included in Table 2, more than 80% analyzed AOH and AME, and around 50% included TEN and TeA, followed by ALT (36%).These were followed by Alternaria toxins ATXs included in 16% of the analyzed studies, whereas only a few publications reported other compounds such as macrosporin (9%), monocerin, ALN, AALs, and infectopyrone (all <3%).Notably, limited information was found about pyrenochaetic acid A (PyA), alternarienonic acid (AIA), and alloTeA and isoALT, recently reported in food samples for the first time [31,105] and metabolites such as AOH3S, AOH3G, AME3S, and AME3G [47,69].In this way, AOH, AME, TeA, and TEN have been shown to occur in food samples frequently [82,97,108], while the occurrence of ALT, isoALT, ATX-I, and AAL toxins is of much lower incidence, mainly due to shortcomings in current analytical methodologies.Slight differences were observed based on the analyzed food matrices.Thus, AOH and AME (both 86%) were the most analyzed ones in cereals and cereal by-products, followed by TEN and TeA (59 and 41%, resp.).Other mycotoxins studied in these matrices were ALN (27%), macrosporin (23%), ALT (14%), and less extent monocerin and infectopyrone (both 5%).The same trend was observed in fruits, vegetables, and derived products, with predominance of AOH and AME (both 79%), followed by TEN (41%), TeA (38%), ALT (34%), and ATXs (14%).

Concluding Remarks
In the last years, several food matrices were analyzed for the presence of Alternaria mycotoxins, with tomatoes and derived products, cereals and cereals by-products, and fruits such as apples and berries as the most representatives.The most common extraction technique was SLE/LLE with several organic solvents mainly ACN, EtOAc, and CH3OH.Other methodologies of relevant importance were QuECh-ERS, dilution-direct injection and SLE-SPE.LC-MS/MS systems were by far the most used analytical techniques for Alternaria mycotoxins determination.AOH and AME, followed by TEN, TeA, and ALT were the most analyzed mycotoxins in food and feed.
With regard to the widespread occurrence of Alternaria mycotoxins in various food and feedstuffs intended for human or animal consumption and their high toxicity, more toxicological studies are needed on the field, transport, storage, and processing stages.The monitoring of their incidence at low concentrations is warranted to find out the extent of human exposure to these contaminants, as well as the influence of cooccurring microorganisms and their produced metabolites on the subsequent enhancement or inhibition of Alternaria species growth.Considerable attention should be paid to the mycotoxin production process in order to take adequate measures for their suppression and decontamination within national and international programs.Moreover, it remains important to examine the potential correlation among these toxic compounds, as different toxicity profiles may occur.Furthermore, the action of purified molecules in various in vitro systems needs to be studied in depth.Fungal population genetics also deserve attention in order to elucidate their role as mycotoxin producers and to understand the Alternaria secondary metabolism (producing parent or conjugated analytes) when subjected to different ecophysiological factors (mainly temperature and water activity), which are able to modulate their toxicological profiles.The use of predictive models to determine the factors that influence Alternaria mycotoxin contamination in the field and across the different processing stages may result in a notable decrease of this contamination throughout the production chain.
Although there are no specific international regulations for any of the Alternaria mycotoxins, EFSA has provided a scientific opinion on the risks for animal and public health related to the presence of Alternaria toxins in food and feed [6].Risk assessment related to food safety is frequently hampered by the lack of quantitative data.Up to now, results on Alternaria mycotoxins risk assessment proved to be inconclusive due to limited representative occurrence and toxicity data.However, application of the threshold of toxicological concern (TTC) approach indicated that there might be a possible risk for human health related to the presence of Alternaria toxins in foodstuffs.Maximum levels admitted for these toxins should be released by EFSA and regulated by the European Union in the near future.As these contaminants could be found in a wide range of food products, their current and future determination is essential for regulatory bodies with the purpose of improving the quality of products and preserving consumers' health.

Figure 1 :
Figure 1: Chemical structure of the main Alternaria mycotoxins.

Table 1 :
Chemical name, molecular formula, molecular weight, and CAS number for the main Alternaria mycotoxins.

Table 2 ,
divided into cereals and cereal by-products (Table 2(a)), vegetables and fruits (Table 2(b)), and other food products (Table 2(c)), describes the analytical method, extraction methodology, analyzed mycotoxins, and limits of detection/quantitation for the analyzed studies.