Detoxification of Mycotoxins through Biotransformation

Mycotoxins are toxic fungal secondary metabolites that pose a major threat to the safety of food and feed. Mycotoxins are usually converted into less toxic or non-toxic metabolites through biotransformation that are often made by living organisms as well as the isolated enzymes. The conversions mainly include hydroxylation, oxidation, hydrogenation, de-epoxidation, methylation, glycosylation and glucuronidation, esterification, hydrolysis, sulfation, demethylation and deamination. Biotransformations of some notorious mycotoxins such as alfatoxins, alternariol, citrinin, fomannoxin, ochratoxins, patulin, trichothecenes and zearalenone analogues are reviewed in detail. The recent development and applications of mycotoxins detoxification through biotransformation are also discussed.


Oxido-Reduction between Alcohols and Ketones
Oxido-reduction between alcohols and ketones of mycotoxins include: (i) oxidation of the hydroxyl group and (ii) reduction of the carbonyl group (Table 2).

Oxido-Reduction between Alcohols and Ketones
Oxido-reduction between alcohols and ketones of mycotoxins include: (i) oxidation of the hydroxyl group and (ii) reduction of the carbonyl group (Table 2).

Substrate
Product Biotransformation System Ref.

De-Epoxidation
De-epoxidation of mycotoxins is commonly found in trichothecene analogues (Table 4) [50,51]. Trichothecene toxicity depends heavily upon the epoxide moiety of the molecule, and opening the epoxide ring dramatically reduces the toxicity [52].

De-Epoxidation
De-epoxidation of mycotoxins is commonly found in trichothecene analogues (Table 4) [50,51]. Trichothecene toxicity depends heavily upon the epoxide moiety of the molecule, and opening the epoxide ring dramatically reduces the toxicity [52].

De-Epoxidation
De-epoxidation of mycotoxins is commonly found in trichothecene analogues (Table 4) [50,51]. Trichothecene toxicity depends heavily upon the epoxide moiety of the molecule, and opening the epoxide ring dramatically reduces the toxicity [52].
Citrinin (CTN/CIT, 57) is a polyketide nephrotoxic mycotoxin commonly present as a natural hazardous contaminant both in food and feed world wide. It was first isolated from the fungus Penicillium citrinum [8]. Dihydrocitrinone (DH-CTN, 58) was detected as the main metabolite of CTN (57) ( Figure S27) in the urine of rats [57] and humans [58]. CTN (57) induced a concentration-dependent increase in micronucleus frequencies at concentrations ≥30 µM, wheas DH-CTN (58) showed no genotoxic effect up to 300 µM. Thus, conversion of CTN (57) to DH-CTN (58) in humans can be regarded as a detoxification step [58].
The marine yeast Kodameae ohmeri was found to transform patulin (PAT, 65) into E-ascladiol (66) and Z-ascladiol (67) through reduction ( Figure S30). High transformation rate was at a temperature of 35 • C and pH between 3 and 6 that indicated the potential application of K. ohmeri for PAT (65) detoxification of the contaminated products [60]. E-ascladiol (66) and Z-ascladiol (67) have been found to exhibit no signs of toxicity towards human cell lines derived from the intestinal tract, kidney, liver and immune system, which demonstrates that PAT (65) detoxification strategies leading to the accumulation of ascladiols should be approaches to limit the PAT (65) risk [61]. When PAT (65) was added in the cell cultures or cell-free supernatant of Lactobacillus plantarum, it was transformed to E-ascladiol (66) and Z-ascladiol (67), which were further transformed into hydroascladiol (68) over a 4-week cell-free incubation at 4 • C ( Figure S31) [62]. Kodameae ohmeri (fungus) [60] E-Ascladiol (66), Z-ascladiol (67), hydroascladiol (68) Lactobacillus plantarum (bacterium) [62] Toxins 2020, 12, x FOR PEER REVIEW 8 of 37 FA (19). The fungus A. tubingensis provides a novel detoxification mechanism against FA (19), which may be utilized to control Fusarium wilt [59]. The marine yeast Kodameae ohmeri was found to transform patulin (PAT, 65) into E-ascladiol (66) and Z-ascladiol (67) through reduction ( Figure S30). High transformation rate was at a temperature of 35 °C and pH between 3 and 6 that indicated the potential application of K. ohmeri for PAT (65) detoxification of the contaminated products [60]. E-ascladiol (66) and Z-ascladiol (67) have been found to exhibit no signs of toxicity towards human cell lines derived from the intestinal tract, kidney, liver and immune system, which demonstrates that PAT (65) detoxification strategies leading to the accumulation of ascladiols should be approaches to limit the PAT (65) risk [61]. When PAT (65) was added in the cell cultures or cell-free supernatant of Lactobacillus plantarum, it was transformed to Eascladiol (66) and Z-ascladiol (67), which were further transformed into hydroascladiol (68) over a 4week cell-free incubation at 4 °C ( Figure S31) [62].

Methylation
Methylation of myctoxins was observed on the hydroxyl groups. The transformation was catalyzed by O-methyltransferase (Table 6).

Methylation
Methylation of myctoxins was observed on the hydroxyl groups. The transformation was catalyzed by O-methyltransferase (Table 6).

Glycosylation and Glucuronidation
Glycosylation/glucuronidation of mycotoxins is the process by which a glucose or glucuronic acid is covalently attached to a hydroxyl group (Table 7). Glycosylation/glucuronidation often increases the polarity of mycotoxins, and reduce their toxicity. Many glycosyltransferases of mycotoxins are present in plants. A UDP-glucosyltransferase involved in the detoxification of deoxynivalenol was revealed from rice (Oryza sativa) [65].
Both 15-monoacetoxyscirpenol (15-MAS, 98) and 4,15-diacetoxyscirpenol (4,15-DAS, 99) were produced by the fungi from the genus Fusarium such as F. sporotrichioides and F. poae [73]. In corn plants, both 15-MAS (98) and 4,15-DAS (99) were, respectively, transformed to 15-MAS 3-glucoside (100) and 4,15-DAS 3-glucoside (101), which were called masked mycotoxins. The structures of transformed products (99 and 100) were deduced on the basis of accurate mass measurements of characteristic ions and fragmentation patterns by using high-resolution liquid chromatography-Orbitrap mass spectrometric analysis. Although their absolute structures were not clarified, 3-OH glucosylation appeared to be the most probable ( Figure S43) [74,75]. As the authors used Fusarium sp. infected corn material, it is not clear whether the corn plant or the fungus itself produced the glucosides just like formation of MAS glucoside [76] as well as the glucosides of HT-2 and MAS [77] produced by Fusarium species.

Esterification
Esterification of mycotoxins is a reaction to form ester mainly from alcohols and carboxylic acids (Table 8). Ochratoxin A (OTA, 21) was converted to OTA methyl ester (110) by the cell cultures of wheat and maize ( Figure S51) [31].

Esterification
Esterification of mycotoxins is a reaction to form ester mainly from alcohols and carboxylic acids (Table 8). Ochratoxin A (OTA, 21) was converted to OTA methyl ester (110) by the cell cultures of wheat and maize ( Figure S51) [31].

Esterification
Esterification of mycotoxins is a reaction to form ester mainly from alcohols and carboxylic acids (Table 8). Ochratoxin A (OTA, 21) was converted to OTA methyl ester (110) by the cell cultures of wheat and maize ( Figure S51) [31].
Ochratoxin C (OTC, 121), which was also called OTA ethyl ester, was hydrolyzed into OTA (21) by deacetylation in rats after oral and intravenous administration ( Figure S59). The very fast conversion of OTC (121) into OTA (21) is a possible explanation of the similar toxicity of ocratoxins A (21) and C (121) to animals and humans [100].

Demethylation
Demethylation of mycotoxins is the process resulting in the removal of a methyl group from a molecule (Table 11). A common way of mycotoxin demethylation is replacement of a methoxyl group by a hydroxyl group. Alternariol 9-O-methyl ether (AME, 8) was converted to alternariol (AOH, 7) with demethylation by the homogenate of porcine liver in the presence of NADPH ( Figure S64) [104].

Demethylation
Demethylation of mycotoxins is the process resulting in the removal of a methyl group from a molecule (Table 11). A common way of mycotoxin demethylation is replacement of a methoxyl group by a hydroxyl group. Alternariol 9-O-methyl ether (AME, 8) was converted to alternariol (AOH, 7) with demethylation by the homogenate of porcine liver in the presence of NADPH ( Figure S64) [104].

Demethylation
Demethylation of mycotoxins is the process resulting in the removal of a methyl group from a molecule (Table 11). A common way of mycotoxin demethylation is replacement of a methoxyl group by a hydroxyl group. Alternariol 9-O-methyl ether (AME, 8) was converted to alternariol (AOH, 7) with demethylation by the homogenate of porcine liver in the presence of NADPH ( Figure S64) [104].

Miscellaneous Reactions
Many other types of biotransformation of mycotoxins have also been reported, such as epimerization, epoxidation, dehydrogenation, dechlorination and their multi-step conversions (Table  13).

Miscellaneous Reactions
Many other types of biotransformation of mycotoxins have also been reported, such as epimerization, epoxidation, dehydrogenation, dechlorination and their multi-step conversions (Table 13).

Detoxification of Aflatoxins
Aflatoxins (AFs) are a group of furanocoumarin mycotoxins mainly produced by Aspergillus species. They are threats to human and animal health, and are classified by the International Agency for Research on Cancer (IARC) as group 1 carcinogens [7,14,125,126]. The furofuran ring of AFs has been recognized as responsible for the toxic and carcinogenic activity upon metabolic activation of the C8-C9 double bond to 8-9 epoxide [7]. The epoxidation is a crucial reaction for carcinogenicity of AFs, since it allows the binding to N7-guanine and the subsequent G to T transversions in the DNA molecule [127]. Activated AFs are also able to form Schiff bases with cellular and microsomal proteins (via methionine, histidine and lysine), thus leading to acute toxicity [128]. The lactone ring also plays a role in AFs toxicity and carcinogenicity. DNA alkylation depends upon both difuranocumarin and lactone moieties of AFs [129].

Detoxification of Aflatoxins
Aflatoxins (AFs) are a group of furanocoumarin mycotoxins mainly produced by Aspergillus species. They are threats to human and animal health, and are classified by the International Agency for Research on Cancer (IARC) as group 1 carcinogens [7,14,125,126]. The furofuran ring of AFs has been recognized as responsible for the toxic and carcinogenic activity upon metabolic activation of the C8-C9 double bond to 8-9 epoxide [7]. The epoxidation is a crucial reaction for carcinogenicity of AFs, since it allows the binding to N7-guanine and the subsequent G to T transversions in the DNA molecule [127]. Activated AFs are also able to form Schiff bases with cellular and microsomal proteins (via methionine, histidine and lysine), thus leading to acute toxicity [128]. The lactone ring also plays a role in AFs toxicity and carcinogenicity. DNA alkylation depends upon both difuranocumarin and lactone moieties of AFs [129].
Many studies have focused on the detoxification of aflatoxins [130]. However, only a few studies detected the converted products and analyzed their toxicity. Two main detoxification pathways, which are modifications of the difuran ring and coumarin structure of AFs, have been investigated. Some reviews on the detoxification of aflatoxins through biotransformation have been available [12,[131][132][133][134].
Among the isolated AFs, aflatoxin B 1 (AFB 1 , 1) is not only the compound with the highest content, but also the most toxic. It is responsible for liver cancer in animals. Microbial degradation of AFB 1 (1) has been well reviewed [130]. For the AFB 1 (1), biotransformation pathways mainly include epoxidation of the carbon-carbon double bond of the furan ring, hydroxylation of the difuran ring, demethylation of methoxy group of aromatic ring, reduction of carbonyl group of cyclopentenone and C3 hydroxylation of cyclopentenone [131,134]. Among them, the reduction of carbonyl group in pantan cycle to hydroxyl group by several fungal species ( Figure S15) [41], and multi-step oxidation-reduction reactions of AFB 1 (1) to AFD 1 (134), AFD 2 (135) and AFD 3 (136) by Pseudomonas putida (Figure S69) [109] were effective reactions for detoxification. The main transformation pathways of AFB 1 (1) are as follows.
(iii) Reduction: Both altertoxin II (50) and stemphyltoxin III (51) with a perylene quinone structure were reduced to alcohols in human colon Caco-2 cells, resulting in the formation of altertoxin I (52) and alteichin (53), respectively. Altertoxin II (50) was also reduced to alcohol in other human cell lines such as HCT 116, HepG2 and V79 [55].

Detoxification of Citrinin
Citrinin (CTN/CIT, 57) is a mycotoxin produced by the fungi from genera Monascus, Aspergillus and Penicillium, which contaminate plant seeds and other foods. CTN (57) is known as a hepata-nephrotoxinc mycotoxin with immunotoxic and carcinogenic properties [8]. The main transformation pathways of CTN (57) are as follows.

Detoxification of Fomannoxin
Fomannoxin (40) is a fungal benzohydrofuran aldehyde phytotoxin, which was produced by the forest pathogenic basidiomycete Heterobasidion annosum during the infection process [43]. Fomannoxin (40) showed growth-inhibiting effects on callus and suspension cultures of conifer cells [44]. The most effective detoxification for fomannoxin (40) was reduction by the cultured cells of Pinus sylvestris ( Figure S17) [44]. The main transformation pathways of fomannoxin (40) are as follows.

Detoxification of Fumonisins
Fumonisins are mainly produced by Fusarium verticillinoids and F. proliferatum, and are structurally similar to sphingolipid long-chain bases such as sphinganine and sphingosine [17]. Fumonisin B 1 (FB 1 , 115) is the most prevalent fumonisin and holds the highest risk for human and animal nutrition among FBs. They contaminate corn and its processed foods [139]. This feature is tightly related to their toxicity mechanism through the inhibition of the sphingolipid biosynthesis in animals, plants, and yeasts [140,141]. A wide variety of diseases in animals such as liver cancer in rats, equine leukoencephalomalacia and porcine pulmonary edema have been associated with fumonisins [142]. They may cause neural tube defects in some maize-consuming populations [143].
Detoxification of fumonisins through biotransformation mainly include hydrolysis with loss of two tricarballylic side-chains by carboxylesterase, and deamination by aminotransferase [90]. The main transformation pathways of fumonisins are as follows.
(ii) Hydrolysis and deamination: FB 1 (115) was hydrolyzed by carboxylesterase with loss of two tricarballylic acid (TCA) groups, followed by deamination by aminotransferase in the presence of pyruvate and pyridoxal phosphate [90].

Detoxification of Patulin
Patulin (PAT, 65), a polyketide lactone, exhibits genotoxic, mutagenic, carcinogenic, teratogenic and cytotoxic properties for humans and animals as well as plants [148,149]. PAT (65) can be produced by various fungal species belonging the genera Penicillium, Aspergillus and Byssochlamys, and particularly, Penicillium expansum is considered as the major producer of PAT (65), which is causative agent of the blue mold disease of stored apples [150]. Detoxification of PAT (65) through biotransformation by using yeast, bacteria and fungi have been shown good results, and it seems to be attractive since it works under mild and environment-friendly conditions [149,151]. If furan or the pyran ring of PAT (65) was destroyed, the transformed products became less or non toxic ( Figures S30, S31 and S82). The main transformation pathways of PAT (65) are as follows.

Detoxification of Trichothecenes
Trichothecenes are mainly produced by the fungi from the genus of Fusarium. Other minor trichothecenes producing species are from the genera of Cephalosporium, Myrothecium, Stachybotrys, Trichoderma and Trichothecium [156][157][158]. Trichothecenes are commonly found in contaminated cereals, particularly in wheat, barley, oats and maize [15].
Trichothecenes are chemically tricyclic sesquiterpenoids, characterized by a double bond at the C-9 and C-10 position, an epoxy functional group at the C-12 and C-13 position and variable numbers of hydroxyl and acetoxy groups. Trichothecenes can be divided into four types (i.e., types A-D) according to characteristic functional groups [159]. Among them, types A and B are of more concern to humans due to their broad and highly toxicity. Data about their natural occurrence in foods are mostly limited to HT-2 toxin (122) (31) does not occur as much as DON (38), but its toxicity is higher than that of DON (38) [156,160].
The double bond between C-9 and C-10, the 12, 13-epoxide ring, and hydroxyl group at C-3 have been considered as the toxicity of trichothecenes in eukaryotic organisms [161]. Removal of these groups results in a complete loss of toxicity [159]. Common toxicity effects of the trichothecenes in humans and animals include diarrhea, vomiting, feed refusal, growth retardation, immunosuppression, reduced ovarian functions/reproductive disorders and even death [162]. At the molecular level, trichothecenes bind to the ribosome, induce a ribotoxic stress leading to the activation of MAP kinases, cellular cell-cycle arrest and apoptosis [160].
Detoxification of trichothecenes via various biotransformation pathways mainly include oxygenation [163], de-epoxidation at the C-12 and C-13 positions [50,164], epimerization of hydroxyl group at C-3 position [113], glycosylation of hydroxyl groups [79] and hydrolysis of acetoxy groups [79,86]. Biotransformation systems include several bacteria and fungi along with their isolated enzymes. The main transformation pathways of trichothecenes are as follows.

Detoxification of Zearalenone Analogues
Zearalenone (ZEN, 33) is a macrocyclic phenolic β-resorcyclic acid lactone. It mainly produced by fungi belonging to the genus Fusarium such as F. graminaearum and F. culmorum. It possesses estrogenic activity in pigs, cattle and sheep [166]. Moreover, alcohol metabolites, such as α-zearalenol (42) and β-zearalenol (43) of ZEN, are also estrogenic and the effects of ZEN (33) and related metabolites on animal reproductive function have been reported [166,167]. ZEN (33) also damages the liver and kidneys and reduces immune function which leads to cytotoxicity and immunotoxicity [12]. The possible pathways available for ZEN (33) biotransformation relate mainly to the hydrolysis of the lactone ring, reduction of the ketonic carbonyl group, modification of the hydroxyl groups (i.e., sulfation and glycosylation), and reduction of the carbon-carbon double bond. The main transformation pathways of ZEN analogues are as follows.
(ii) Reduction of the carbon-carbon double bond: ZEN (33) was transformed to zearalanone (44) with reduction of the carbon-carbon bond in ovine [49].

Conclusions and Future Perspectives
This review described detoxification of mycotoxins through biotransformation by using bacteria, fungi, plants and animals, as well as the isolated enzymes. As can be seen from the examples given in this review that both organisms and the isolated enzymes possess considerable biochemical potentials to convert mycotoxins. The recently-reported examples, coupled with the emergence of some efficient commercialized biological/enzymatic agents, highlight the promise of this approach to address the safety of animal feed and human food [11,168,169].
The reaction types and stereochemistry of conversion depend on the functional groups in the molecular structures of mycotoxins together with the specific enzymes provided by the organisms. Detoxification through biotransformation is now an important strategy to eliminate mycotoxins from food and feed. Indeed, not all converted products are nontoxic. Some converted products are still toxic or even more toxic [170,171]. Some mycotoxins are converted into the masked forms such as ZEN 14-O-glucoside (107) and ZEN 16-O-glucoside (108), which can be accumulated in organisms and cannot be eliminated [81,172,173]. Through the studies of structure-activity relationship (SAR), the essential functional groups for toxicitiy of mycotoxins have been clarified [7,161,174]. This should be the focus of the research on mycotoxin biotransformation for detoxification in the coming years.
Bacteria and fungi that can transform mycotoxins to less toxic or nontoxic products serve as a source of enzymes that can be used to decontaminate agricultural commodities or used as feed additives. More and more degradation enzymes of mycotoxins have been purified and identified from microorganisms [7,175]. The corresponding genes have been cloned and expressed in the engineered microorganisms. Both purified enzymes and engineered strains have potential applications for mycotoxin degradation in food and feed industry. Some genes that control the detoxifications of mycotoxins, such as the genes of trichothecene acetyltransferase (TRI101) [176] and the zearalenone lactonohydrolase (zhd101) [177], have been cloned and expressed in plants to limit pre-harvest contamination of crops.
In plants, sequential hydroxylation, glycosylation and demethylation of fungal phytotoxins can avoid plant cell death and overcome the fungal invader [24]. Detoxification of fungal phytotoxins through biotransformation by plants should be an important plant defensive mechanism against fungal pathogens [25].
In short, detoxification of mycotoxins through biotransformation provides a reliable reference strategy for the management of mycotoxins in foods and feeds. It will help us to better understand the fate of mycotoxins in animals and humans, as well as to provide basic information for the risk assessment of mycotoxins for food and feed safety. Further investigations, especially the development of methods to utilize the multi-reaction processes as well as to clone and express the genes of detoxification enzymes in organisms, will be necessary for the detoxification of mycotoxins through biotransformation.