Keywords

1 Introduction

Nivalenol (NIV) was discovered by Japanese scientists from the culture medium of Fusarium nivale strain Fn-2B obtained from Fusarium head blight-infected wheat, in Japan [1]. Subsequently, Fn-2B was reclassified as a new species, F. kyushuense O’Donnell & T. Aoki [2] from molecular phylogenetic analyses. Other Japanese researchers identified the chemical structure of NIV, fusarenon-X (4-acetyl NIV), and deoxynivalenol (DON) [3–6].

Trichothecene mycotoxins are the main mycotoxins produced by Fusarium species. Although there are macrocyclic and non-macrocyclic mycotoxins, the latter primarily contaminate wheat, barley, and maize and are classified into two types: Type A, including T2 toxin and HT2 toxin, diacetoxyscirpenol, and neosolaniol and type B, including DON, NIV, and 4-acetyl NIV. T2 toxin is reportedly the causative agent of outbreaks of foodborne diseases that occurred in the Orenburg region of the USSR during the 1930s–1940s. This outbreak was termed alimentary toxic aleukia (ATA), and symptoms of this disease include nausea, emesis, diarrhea, leukopenia, hemorrhages, and shock-mediated death.

Although the outbreak caused by type B trichothecenes is less severe than ATA, acute human illnesses caused by the consumption of Fusarium-infected wheat and barley have been reported in Japan, India, and China [7–9]. In these outbreaks, DON, NIV, and zearalenone were commonly detected in food.

Concerning type B trichothecenes, chronic and acute adverse health effects are of considerable concern. DON and NIV have been reported to suppress the immune system, depending on the dose and frequency of exposure [10].

Compared to DON, the distribution of NIV-producing fungi is limited and lacks toxicological and exposure data. DON has been evaluated by the FAO/WHO Joint Expert Committee of Food Additives while NIV has not. In countries where NIV contaminates cereals, the adverse health effect induced by NIV is a serious problem, and NIV is considered to be one of the mycotoxins whose risk needs to be assessed and regulated. In Europe, a large-scale surveillance of trichothecenes in food from 2000 to 2002 revealed the occurrence of NIV contamination [11]. In Japan, as NIV-producing fungi also occur, the occurrence of DON and NIV were surveyed in food. Risk assessment has been completed by the Food Safety Commission (FSC) of Japan. The European Commission’s (EC) (now the European Union, or EU) Scientific Committee on Food (SCF) evaluated and determined a provisional daily tolerable intake as 0.7 μg/kg of body weight (bw) [12], but the FSC of Japan concluded that this level should be 0.4 μg/kg of bw.

This chapter reviews the latest findings on the mycology, biosynthesis, occurrence, toxicology, analytical methods of analysis, and risk assessment of NIV.

2 General Biology

2.1 Common/Systematic Name

  • CAS (No.23282-20-4)

  • 3α, 4β, 7α, 15-tetrahydroxy-12, 13-epoxytrichothec-9-en-8-one, IUPAC

  • 12, 13-epoxy-3α, 4β, 7α, 15-tetrahydroxytrichothec-9-en-8-one

2.2 Molecular Formula/Molecular Weight

C15H20O7: 312. 32

Nivalenol

2.3 General Characteristics

Crystals from methanol; mp., 80–90 °C; dried in presence of P2O5 in reduced pressure; mp., 222–223 °C, tetraacetate; mp., 168–170 °C, [α] D 24+ 21.54° (c = 1.3, in EtOH)

2.4 Spectral Data

λ↓Max ↑MeOH 218 Nm (Ɛ = 7,500), Tetraacetate, 227 Nm (Ɛ = 7,900)

3 Mycology

Fusarium head blight (FHB) is a harmful disease that infects wheat, barley, and other cereals. This disease not only reduces grain yield and quality but also causes the contamination of trichothecene mycotoxins such as DON and NIV and other mycotoxins in the grain. The pathogens of FHB, Fusarium species, can be classified into two chemotaxonomic groups, the DON chemotype and the NIV chemotype [13–15]. Lee et al. [14] clarified that a single gene (Tri13) is responsible for the differential ability to produce DON or NIV. Fusarium DON chemotypes are found worldwide, while NIV chemotypes are found in more restricted regions, namely, limited areas of Asia, Africa, Europe, and North America [16, 17]. F. kyushuense and F. graminearum complex species (F. asiaticum, F. culmorum, F. crookwellense, F. equiseti, and F. poae) are also reported to produce NIV (Table 100.1).

Table 100.1 NIV-producing fungi
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The F. graminearum species complex [F. graminearum Schwabe; teleomorph: Gibberella zeae (Schwein.) Petch] is a species complex consisting of at least nine biogeographically structured lineages based on molecular phylogenetic analyses using worldwide collections. The lineages are as follows: lineage 1 is F. austroamericanum, lineage 2 is F. meridionale, lineage 3 is F. boothii, lineage 4 is F. mesoamericanum, lineage 5 is F. acacia-mearnsii, lineage 6 is F. asiaticum, lineage 7 is F. graminearum s. str., lineage 8 is F. cortaderia, while lineage 9 is F. brasilicum. On potato dextran agar (PDA), the F. graminearum species complex produces abundant white mycelia that become yellow to brownish or rose-colored as the cultures age (for 2 weeks); the color of the bottom surface of the colony is usually deep red. The undersurface is usually carmine red (Fig. 100.1). Macroconidia are usually long, slender, and slightly curved to straight, with five to six septa and a well-developed foot cell. Microconidia are absent; chlamydospores are rare but may form in macroconidia [18].

Fig. 100.1
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Fusarium graminearum. (a): giant colony on PDA (reverse side) (b) Macroconidia

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Trichothecene chemotypes of the F. graminearum species complex consist of three production groups according to strain differences: DON and 3-acetyl deoxynivalenol (3ADON), DON and 15-acetyl deoxynivalenol (15ADON), and NIV [13–15]. In the F. graminearum species complex, lineage classification is not well correlated with the trichothecene chemotype [19, 20].

Regional mycological studies discovered that geographic differences exist among these trichothecene chemotypes [21, 22]. The 3ADON productive group was not detected in 15 strains of F. graminearum s. str., and all 13 strains of F. cortaderia in New Zealand were of the NIV production group [23]. The NIV production group in F. asiaticum has been identified and is likely to represent about 25 % of the population of the F. graminearum species complex in Louisiana, USA [24]. In Japan, F. graminearum s. str. is predominant in the northernmost island (Hokkaido), while in southern areas, F. asiaticum is predominant [25].

In terms of pathogenicity, DON-producing strains are more aggressive and virulent than NIV producers [26, 27]. However, the NIV production group in F. asiaticum isolated from the western part of Japan was significantly more virulent than the most virulent DON chemotype F. graminearum s. str. strains [28].

Fusarium culmorum (W. G. Smith) Sacc. is the second most important FHB pathogen in wheat and strains of both DON and NIV producers exist. F. culmorum inhabits cooler areas such as North, Central, and Western Europe; North America; and Eastern Australia. Strains of both the DON and NIV production groups were isolated from England and Wales: in the south and west of England and Wales, NIV chemotypes are predominant, whereas in the north and east, DON chemotypes are predominant [29].

Fusarium poae (Peck) Wollenw. exists in Europe, North America, and Japan. It is one of the causative fungi causing FHB in small-grain cereals. In the northernmost area of Japan, the NIV production group in F. poae plays a role in the contamination of grains with NIV [30]. The NIV production group of F. poae was frequently found in Sweden, and NIV contamination has become a considerable concern in Scandinavia [31].

Fusarium crookwellense L.W. Burgess, P. E. Nelson & Toussoun was first isolated in Australia in 1971 as a new species [32]. It is another FHB pathogen found in small-grain cereals in Poland, New Zealand, China, Canada, Japan, and other countries [33]. F. crookwellense isolated from scabby wheat in the northernmost area of Japan produced NIV, 4-acety NIV, and zearalenone (ZEN) when cultured on rice-based medium [30].

4 Biosynthesis

4.1 History of Trichothecene Biosynthesis Studies

In earlier days, biosynthesis studies of trichothecenes were initiated using Trichothecium roseum as a model [34]. Having elucidated the scheme of biosynthesis from farnesyl pyrophosphate (FPP; 1) (see Fig. 100.2), via a cyclized product trichodiene (TDN; 2), to trichothecin [35, 36], researchers have moved to study Fusarium species that produce agriculturally important trichothecenes, such as T-2 toxin, DON, and NIV. Feeding experiments with TDN demonstrated its precursor role in DON biosynthesis [37], as was the case of T. roseum. Other postulated intermediates in the biosynthetic pathway were thereafter proven by blocked mutant analysis, precursor feeding experiments, and molecular genetic approaches using Fusarium species, including F. culmorum, F. graminearum, and/or F. sporotrichioides [38–40]. Compared to trichothecenes of other fungal genera, Fusarium trichothecenes are distinguished by the presence of a hydroxyl or O-acetyl at C-3.

Fig. 100.2
figure 01352

A common pathway to ITD in the biosynthesis of Fusarium trichothecenes. These early steps are conserved between type A (e.g., T-2 toxin) and type B (e.g., NIV) trichothecene producers. Tri genes that are responsible for these steps are indicated below the half-brackets in red letters. Functional groups highlighted in red indicate that they appeared as the results of expression of these Tri genes. Oxygen atom involved in intramolecular attack to C-11 in the nonenzymatic cyclization is shown in blue

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4.2 Formation of 12, 13-Epoxytrichothec-9-ene Skeleton (Trichothecene Skeleton): Early Stage of Biosynthesis

In the biosynthesis of Fusarium trichothecenes, TDN is oxygenated by a cytochrome P450 monooxygenase (CYP) in the following order: 2α-hydroxylation, 12, 13-epoxidation, 11α-hydroxylation, and 3α-hydroxylation (Fig. 100.2). The last oxygenation step proceeds only in Fusarium species, which makes Fusarium trichothecenes unique among all others (lacking a C-3 substituent) of non-Fusarium origin. Under acidic conditions, the resulting fully oxygenated intermediate, isotrichotriol (3), appears to undergo second cyclization nonenzymatically to give isotrichodermol (4) [41], the first trichothecene intermediate with a toxic 12,13-epoxytrichothec-9-ene skeleton (for a comprehensive review, see ref. [42]). Although isotrichodermol was not isolated as a natural product from wild-type Fusarium strains in the biosynthesis studies of many research groups, this may not be unreasonable in view of the importance of 3-O-acetylation in trichothecene biosynthesis; perhaps, isotrichodermol must readily be converted to isotrichodermin (ITD; 5) (see Fig. 100.2) for self-protection of the trichothecene-producing fusaria [43]. ITD, a natural product isolated from a wild-type strain, is further metabolized to various important Fusarium trichothecenes [44].

4.3 Molecular and Genetic Studies of F. sporotrichioides as a Basis to Understand NIV Biosynthesis

In 1989, the first isolation and characterization of trichothecene biosynthetic gene was reported for T-2 toxin-producing F. sporotrichioides using an antibody raised against a purified enzyme [45]; this gene, designated Tri5 (formerly Tox5 in the literature), is responsible for the first committed step in the biosynthesis. Since cosmid clones containing Tri5 complemented the tri3 – and tri4 – blocked mutants, but not the tri1 – mutant, at least some of the trichothecene biosynthetic genes (Tri genes) proved to be clustered around Tri5 [46]. On the basis of this finding, other Tri genes, including Tri3 and Tri4, were identified on this core gene cluster. Three additional Tri genes necessary for T-2 toxin biosynthesis, Tri101 alone and Tri1 and Tri16 adjacent to each other, occur separated from the core gene cluster (for reviews, see ref. [42, 47]). Roles of the Tri genes in T-2 toxin biosynthesis were examined by molecular genetic approaches, including targeted gene disruption and heterologous gene expression. The functions of F. sporotrichioides pathway Tri genes (FsTri genes) are summarized as follows (see Figs. 100.2 and 100.3):

  1. 1.

    Tri5 (encoding trichodiene synthase). TRI5 catalyzes cyclization of all-trans-FPP via nerolidyl pyrophosphate to TDN [36, 45].

  2. 2.

    Tri4 (encoding a multifunctional CYP responsible for conversion of TDN to isotrichotriol). Fusarium TRI4 [48], which is grouped into a CYP58 family, catalyzes four consecutive oxygenation steps from TDN to isotrichotriol as follows: TDN → 2α-hydroxytrichodiene → 12,13-epoxy-9,10-trichoene-2α-ol → isotrichodiol → isotrichotriol [49].

  3. 3.

    Tri101 (encoding trichothecene 3-O-acetyltransferase). TRI101 catalyzes conversion of isotrichodermol to ITD [50]. Different from other pathway Tri genes, Tri101 was first cloned from F. graminearum as a gene that confers resistance to T-2 toxin; subsequent analysis with F. sporotrichioides revealed its orthologue in the region of synteny [51]. In addition to isotrichodermol, many trichothecenes, including DON, T-2 toxin, and NIV, serve as good substrates of TRI101 [42]. In the amino acid sequence of TRI101, consensus sequences of acetyltransferases, HXXXDG and DFGWGKP, are found [50]. Apart from its role of self-protection against trichothecenes, C-3 acetyl is essential to serve as substrates of the enzymes in later steps in the biosynthesis.

  4. 4.

    Tri11 (encoding ITD C-15 hydroxylase). TRI11, the first member of a new CYP family CYP65A1, catalyzes hydroxylation of ITD to give 15-deacetylcalonectrin (15-deCAL; 6) [52].

  5. 5.

    Tri3 (encoding 15-deCAL 15-O-acetyltransferase). TRI3 catalyzes 15-O-acetylation of 15-deCAL to give calonectrin (CAL; 7) [53]. Other trichothecenes also serve as good substrates of TRI3. However, compared to 3-acetyltrichothecenes, 3-hydroxytrichothecenes are rather poor substrates [54]. The two consensus sequences of acetyltransferases described in the above section (3) were conserved in TRI3 as was the case with TRI101.

  6. 6.

    FsTri13 (encoding 3-acetyltrichothecene C-4 hydroxylase). In T-2 toxin biosynthesis, FsTRI13 catalyzes hydroxylation at C-4 of CAL [55]. In addition to CAL, other trichothecenes with functional groups at C-8 (and also at C-7) serve as good substrates of FsTRI13.

  7. 7.

    Tri7 (hypothesized to encode 3-acetyltrichothecene 4-O-acetyltransferase). TRI7 is involved in 4-O-acetylation of 3, 15-diacetoxyscirpenol (3, 15-DAS; 8) and its derivatives in T-2 toxin biosynthesis [56]. However, different from TRI101 and TRI3, TRI7 does not possess the consensus sequences conserved among acetyltransferase family; TRI7 shows no amino acid sequence similarity to any other proteins reported so far. Attempts to prepare recombinant TRI7 were not successful, and its enzymatic function has not yet been rigorously proven.

  8. 8.

    FsTri1 (encoding 3-acetyltrichothecene C-8 hydroxylase). FsTRI1 mainly catalyzes hydroxylation of 3, 4, 15-triacetoxyscirpenol (3, 4, 15-TAS; 9) (and to a lesser extent CAL) to give 3-acetylneosolaniol (3-ANEO; 10) (and 8-hydroxycalonectrin) in T-2 toxin biosynthesis [57]. FsTRI1 shows broad substrate specificities and also accept ITD (5) and 3, 15-DAS as substrates. The broad substrate specificity of FsTRI1 and FsTRI13 allow branching of the biosynthetic pathway after CAL (7) in T-2 toxin biosynthesis (Fig. 100.3).

  9. 9.

    FsTri16 (encoding an acyltransferase necessary for formation of ester side chain groups at C-8). FsTRI16 mainly catalyzes esterification at C-8 of 3-ANEO to give 3-acetyl T-2 toxin (11) [58].

  10. 10.

    Tri8 (encoding trichothecene deacetylase). TRI8 removes an acetyl from C-8 of the trichothecene skeleton in T-2 toxin biosynthesis [59].

Fig. 100.3
figure 01353

Biosynthetic steps to F. sporotrichioides T-2 toxin and F. graminearum NIV. Only major pathways to each trichothecene molecule are indicated, and minor pathways are not illustrated. Tri genes that are responsible for these steps are indicated in red letters. Functional groups highlighted in red indicate that they appeared as the results of expression of these Tri genes

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In addition to the pathway Tri genes, the core gene cluster contained two regulatory Tri genes and one transporter Tri gene, which are important for T-2 toxin production: Tri6 encoding a zinc fingerlike transcription factor [60], Tri10 encoding a novel protein with a role of other Tri gene activation [61, 62], and FsTri12 encoding a trichothecene efflux pump [63]. FsTri12 is indispensable for a high level production of T-2 toxin.

The structure of the core gene cluster and the function of the Tri genes were conserved between F. sporotrichioides and F. graminearum [14, 55, 56, 64, 65]. In this way, the identification of FsTri genes served as a useful tool for molecular biological analysis of F. graminearum that produce type B trichothecenes [66].

4.4 Biosynthesis of NIV

Compared to the structure of T-2 toxin, NIV is characterized by the presence of a keto at C-8 and a hydroxyl at C-7; also, C-4 and C-15 are not acetylated, and instead, hydroxyls are attached to these positions (see Fig. 100.3). To elucidate the biosynthetic pathway of NIV, FgTri genes in the core gene cluster were isolated on the basis of nucleotide sequence similarity, and their functions were characterized by the molecular approaches. As to the homologues of FsTri1 and FsTri16 that occur outside the core genes cluster, their sequences shared similarity only at the amino acid sequence level; Tri1 is much more divergent between these Fusarium species (viz., 59 % identity between FsTRI1 and FgTRI1), and Tri16 was found as a pseudogene adjacent to FgTri1 [67]. The results of targeted gene disruption and heterologous gene expression suggested that most of the enzymes encoded by the FgTri genes show the same features as those of F. sporotrichioides. However, the following two enzymes exhibit significant differences in their activities depending on their origin:

  1. 1.

    Compared to FsTRI13, the substrates of FgTRI13 appear to be limited to a group of trichothecenes that have a hydroxyl at C-7 and/or a keto at C-8.

  2. 2.

    FgTRI1, but not FsTRI1, catalyzes oxygenation at both C-7 and C-8 of 4-deoxytrichothecenes [68].

Together with the analyses of other FgTri genes, a major biosynthetic pathway to NIV is hypothesized to proceed as illustrated in Fig. 100.3: ITD → 15-deCAL → CAL → 7,8-dihydroxycalonectrin (DHC, 12) → 3,15-diacetyldeoxynivalenol (3,15-diADON, 13) → 3,15-diacetylnivalenol (3,15-diANIV, 14) → 3,4,15-triacetylnivalenol (3,4,15-triANIV, 15) → 4,15-diacetylnivalenol (4,15-diANIV, 16) → 4-acetylnivalenol (4-ANIV, 17) → NIV (18). In this scheme, all the biologically acetylatable position, namely, C-3, C-15, and C-4, are once acetylated in this order in the biosynthesis and then deacetylated at a later stage. Recently, a gene responsible for deacetylation at C-4 of 4-ANIV was identified and characterized. This gene, designated Tri104, is separated from all other known Tri genes in the genome of F. graminearum (our unpublished results).

5 Toxicology

5.1 Absorption, Distribution, Metabolites, and Excretion

NIV is mainly absorbed from the intestine, and 11–48 % of administered NIV remains in human bodies until 7.5 h post administration [69]. Acetyl NIV is immediately converted into NIV in serum after intravenous and oral administration. The bioavailability of acetyl NIV was 9.8 % and 19.5 % in broiler chickens and ducks, respectively [70].

In vivo examination using healthy swine showed that NIV was mainly absorbed from the ileum [71]. In vitro examination using a Caco-2 cell line (human intestinal epithelial cell line) showed that apical to basal transportation of NIV was simple diffusion depending on energy [72]. The maximum concentration of 3H-labeled NIV and acetyl NIV was detected in serum after 60 min and 30 min in female ICR mice. The area under the curve of acetyl NIV was 10-fold higher than that of NIV. Acetyl NIV is metabolized in the liver and kidney [72].

NIV is converted by intestinal flora bacteria into less toxic de-epoxy NIV in the intestine as is DON. When NIV was anaerobically cultured with swine feces in vitro, NIV was converted into epoxy NIV. Swine that lacked de-epoxidation ability acquired this ability after their faeces with known de-epoxidation ability (containing as yet unclassified intestinal flora bacteria with de-epoxidation ability) were spread out in pens [73]. When NIV and stomach juice of bovine lumen were anaerobically cocultured in vitro, about 80 % of NIV was converted into de-epoxy NIV [73]. NIV and acetyl NIV are distributed in the serum, liver, kidney, and placenta [74]. Eighty percent of administered NIV was excreted in feces as de-epoxy NIV, while 1 % was excreted in urine as NIV in Wistar rats [75]. In chickens, trace levels of NIV were detected in liver and bile, while NIV and de-epoxy NIV were excreted through the feces in more than 10 % of administered NIV [76]. NIV and acetyl NIV were transmitted to milk in lactating mice [74].

As shown in Fig. 100.4, NIV is metabolized by intestinal flora into de-epoxy NIV and then excreted to urine and feces, while acetyl NIV is converted into NIV in serum and organs.

Fig. 100.4
figure 01354

Metabolite pathway of NIV

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5.2 Acute Toxicity

Acute toxicity of NIV is shown in Table 100.2.

Table 100.2 LD50 of NIV after oral administration
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In 6-week-old male ddY mice, the 50 % lethal dose (LD50) of NIV was 38.9 mg/kg of bw after oral administration, 7.4 mg/kg of bw after intraperitoneal injection, 7.2 mg/kg of bw after subcutaneous injection, and 7.3 mg/kg of bw after intravenous injection. Mice died within 3 days due to the effusion of blood and congestion in the intestine. In 5-week-old F344 rats, the LD50 of NIV was 19.5 mg/kg of bw/os and 0.9 mg/kg of bw after subcutaneous injection. Diarrhea and congestion in the gastrointestine were observed. In duck, vomiting was observed after 1.0 mg/kg of bw of NIV, and 0.4 mg/kg of bw of acetyl NIV was administered by subcutaneous injection [79].

5.3 Short-Term Studies of Toxicity

Short-term studies of toxicity were performed in mice, rats, pigs, and chickens, as summarized in Table 100.3.

Table 100.3 Short-term studies of NIV
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5.3.1 Mice

C57BL/6 mice were given moldy rice containing at 0, 5, 10, and 30 mg/kg of NIV for 24 days. At 30 mg/kg of NIV, a significant reduction in red blood cell number was observed, but organ weight and the rate of weight gain did not change [80]. When using a diet containing NIV-contaminated rice at 0, 6, 12, and 30 mg/kg for 4 or 12 weeks, weight gain was inhibited in a dose-dependent manner. From these results, LOAEL was estimated to be 0.7 mg/kg of bw [82].

5.3.2 Rats

When Sprague-Dawley rats were treated with diets containing NIV at 0, 6, and 12 mg/kg for 2 or 4 weeks, a significant reduction in feed intake was observed after 1–2 weeks in the group given 0.6 mg/kg of NIV. However, rats recovered after 4 weeks. The low-observed-adverse-effect level (LOAEL) was estimated at 0.6 mg/kg of bw based on the reduction in organ weight [83]. Toxicity studies of single oral administrations using male and female F344 rats showed sedation, eyelid closure, staggering gait, diarrhea, and congestion of the lung and digestive tract. The oral LD50 value was estimated at 19.5 mg/kg in both sexes in a repeat oral test, purified NIV was given orally at daily doses of 0.4 and 2.0 mg/kg of bw for 30 days. Takahashi et al. reported subchronic toxicity using F344 rats fed a diet containing 0, 6.25, 25, or 100 ppm of pure NIV for 90 days. A decrease in bw and loose stools were observed at 100 ppm. In both sexes, bw was also reduced at 25 ppm in males from 6 weeks. In a hematological observation, the white blood cell count decreased after exposure to 100 ppm in males and to 6.25 ppm in females. Based on hematological data, the LOAEL of NlV was determined to be less than 6.25 ppm (corresponding to 0.4 mg/kg of bw/day for both males and females) [86].

5.3.3 Pigs

After pigs were exposed to purified NIV, no feed refusal, vomiting, or change in clinical appearance occurred, but a macroscopic examination showed gastrointestinal erosion and signs of nephropathy at low doses. Exposure to a high dose of NIV decreased the number of spleen cells. Histological data indicated that exposure of pigs to NIV in the diet caused pathological changes in the kidneys and gastrointestinal tract and reduced the number of splenocytes [87].

5.3.4 Chickens

When male broiler chickens were exposed to feed containing NIV, there was an increase in the uric acid concentration in serum, reduction in the rate of weight gain, gastrointestinal erosion, duodenum internal bleeding, swollen cloaca, and oviducts with immature eggs [76].

5.4 Chronic Studies and Carcinogenesis

Two long-term studies have been reported by the same Japanese group. In both studies, moldy rice powder containing NIV was used as the diet. This moldy rice was estimated to contain 3,147 mg/kg of NIV but no fusarenon-X. The first was a 1-year feeding study in which female C57BL/6CrSlc (SPF, 7-week-old mice) were given diets containing 0, 6, 12, and 30 mg/kg NIV; bw gain and feed efficiency showed a dose-dependent correlation. No changes were observed in the liver, thymus, spleen, kidneys, stomach, adrenal glands, pituitary gland, ovaries, sternum, bone marrow, lymph node, brain, and small intestines with or without Peyer’s patch portion. Leukopenia was observed in the group administered 30 mg/kg NIV after 6 months and in all NIV-treated groups after 1 year. The LOAEL was determined to be 6 mg/kg of diet (corresponding to 0.68 mg/kg of bw) [77]. The other report was a 2-year feeding study in which the feeding conditions were identical to the 1-year feeding study. A reduction in bw gain was observed in all treated groups of animals. In the group given 30 mg/kg of NIV, leukopenia was observed, but it was not statistically significant. No tumors were found in any of the treated groups. Compared with the ratio of naturally occurring tumors, there was no difference between the treatment group and the control group. The LOAEL was 6 mg/kg feed (corresponding to 0.66 mg/kg of bw) [89] (Table 100.4).

Table 100.4 Long-term studies of toxicity
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5.5 Genotoxicity

Table 100.5 shows a summary of the results of genotoxicity studies of NIV. Some in vitro studies have been reported (Table 100.5A) using V79-E cells (a Chinese hamster lung-derived cell line), NIV induced cell cycle retardation. In the presence of metabolic activation (S9 mix), slight chromosomal aberrations were seen. These effects were nonspecific, suggesting that they were caused by inhibited protein synthesis [89].

Table 100.5 Genotoxicity studies of NIV
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In a chromosome aberration test using V79 cells, NIV purified from contaminated corn and barley induced, at 0.001–0.03 μg/mL and 0.03 μg/mL, respectively, a two to three-fold increase in chromosomal aberrations compared with the control [91, 92]. In a short-term transformation assay using v-Ha-Ras-transfected BALB/3 T3 cells, NIV showed no initiation or promotion activity [96]. A single-cell gel electrophoresis (comet) assay of NIV was conducted using CHO cells and ICR mice (4 males/group). At 50 and 100 μg/mL, NIV damaged the DNA of CHO cells in the absence of a metabolic activation system [92].

In an in vivo comet assay (Table 100.5B), oral treatment with NIV (20 mg/kg bw) resulted in DNA damage in the kidneys, bone marrow, stomach, jejunum, and colon. After intraperitoneal administration of NIV, no DNA damage was observed except in the colon [92].

5.6 Immunotoxicity

5.6.1 Effects on Immune Responses

As many scientists have indicated, NIV stimulates or suppresses the immune system depending on the dose as well as the presence of DON. Oral administration of 10 and 15 mg of NIV induced apoptosis in CD4(+) and CD8 (+) cells in thymus, Peyer’s patch, and spleen in a dose-dependent manner [93, 94]. The effect of NIV on susceptibility against infectious diseases was shown by an in vivo infection experiment using BALB/c mice in which NIV at 6 mg/kg had no effect on survival rate against Salmonella infection [95]. However, in an in vitro experiment using RAW 264.7 cells, NIV inhibited the transcription activity and expression of inducible NO synthase (iNOS) by lipopolysaccharide (LPS) [96] (Sugiyama 2010).

5.6.2 Changes in Serum IgA Levels and IgA Nephropathy

Increasing IgA and induction of IgA nephropathy by NIV have been reported. These effects have been observed in mice but not in rats ([84], Table 100.6).

Table 100.6 Effect of NIV on IgA production
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After C57BL/6 mice (ten males/group) were treated three times/week for 4 weeks by oral gavage with NIV (solvent: 5 % gum arabic water solution), the highest dose in that experiment (8.870 mg/kg bw) group showed a significant increase in plasma IgG but no changes in IgA [81], but 0.071 mg/kg bw/day and higher dose groups (0.355 mg/kg bw/day) showed a significant increase in plasma IgA [98]. When C3H/HeN, C3H/HeJ, and BALB/c mice (9–12 females/group) were treated for 4 or 8 weeks with feed containing 0, 6, or 12 mg/kg feed (corresponding to 0, 0.9, or 1.8 mg/kg bw/day, respectively) of purified NIV, the NIV-treated groups demonstrated an increase in IgA accumulation in glomeruli and increased serum IgA, particularly in the 12 mg/kg feed dose group at 8 weeks [99]. In a single oral administration study, Peyer’s patches of BALB/c mice given NIV at 0 or 15 mg/kg bw showed a significant increase in all B cell subpopulations, particularly IgA + B cells, with the numbers of IgA + and IgM + B cells remaining higher than those of the control group [100] (#649).

Interestingly, an experiment using ovalbumin-TCR Tg (OVA-specific T cell receptor transgenic) mice (4 males/group) indicated that NIV significantly inhibited total IgE production and OVA-specific IgE, IgG1, and IgA production [101]. In F344 rats, NIV increased IgM level significantly but not IgG and IgA level even in the group given an oral dose of 6.9 mg/kg bw/day [84].

In pigs fed with purified NIV at 0, 2.5, or 5 mg/mg in feed for 21 days, no significant differences were observed in plasma IgA levels between the control and treatment groups [87].

6 Other Toxicities

NIV inhibits protein and DNA synthesis but not RNA synthesis [102]. NIV inhibited protein synthesis in rabbit reticulocytes with an IC50 value of 6 μg/mL [103]. Cytotoxicity of NIV, 4-acetyl NIV, and de-epoxy NIV on 3 T3 cell growth was IC50 1.19 ± 0.06 mM (373 ± 20 ng/mL), 0.72 ± 0.04 mM (255 ± 13 ng/mL), and 64.2 ± 3.14 mM (19030 ± 930 ng/mL), respectively [104].

7 Outbreaks Associated with NIV

Outbreaks linked with Fusarium-contaminated cereal-based foods occurred in Japan and Korea during the 1940s–1960s [9]. The symptoms reported were nausea, diarrhea, and emesis. In China, more than 30 gastroenteritis outbreaks from 1961 to 1981 were associated with the consumption of “scabby,” i.e., Fusarium-infected wheat, barley, or maize [8]. Trichothecenes were predicted to be the causative agent. An outbreak of trichothecenes occurred in the Kashmir Valley, India, during June to September 1987 in which 50,000 people suffered from gastrointestinal disorders [105, 106]. The cause of the outbreak was assumed to be the consumption of bread made from mold-damaged wheat, evidenced by the presence of molds such as Fusarium sp. and Aspergillus sp. and varying quantities of trichothecene mycotoxins. The concentration of DON, acetyl DON, NIV, and T-2 toxin in causative wheat was 0.34–8.4, 0.6–2.4, 0.03–0.1, and 0.55–4 mg/kg, respectively [105, 106].

8 Analytical Methods

Since NIV occurs as a co-contaminant with other trichothecene mycotoxins, it is often analyzed simultaneously with the co-contaminants rather than alone. Analytical methods developed so far include thin layer chromatography (TLC); capillary gas chromatography (GC) with electron-capture detection (ECD), flame ionization detection (FID), or mass spectrometric detection (GC/MS); high-performance liquid chromatography (HPLC) with ultraviolet (UV), fluorescence, or mass spectrometric detection; supercritical fluid chromatography (SFC); and time-of-flight mass spectrometry (LC/TOF-MS).

Usually, to analyze trichothecenes in foods and feeds, solvent extraction is essential. Aqueous methanol and acetonitrile are commonly used for extraction. For applying GC–MS, HPLC, and LC–MS/MS, a sample needs to be cleaned up with a charcoal-alumina-celite, florisil, silica gel, or solid-phase extraction column. For laboratory experiments, TL is very useful because of its low cost and simplicity. However, for surveillance studies, trichothecene mycotoxins coexist with other trichothecenes, and a simultaneous analytical method for the determination of some trichothecenes and Fusarium toxins is considered to be more practical than a single method. A decade ago, GC was very popular to analyze some trichothecene mycotoxins in food, but for GC analysis, various derivatives are needed that are sometimes troublesome.

GC is typically used conventionally for simultaneous analysis for trichothecene mycotoxins and ZEN but requires trimethylsilyl derivatization before analysis. GC–FID produced good results in a validation of the EU Standards, Measurements and Testing Programme [107]. However, the GC method requires a derivatization procedure, which generally causes a loss of time and recovery.

On the other hand, LC using UV requires no derivatization procedure [108–111]. HPLC–UV (220 nm) for the GC–MS determination of DON and NIV has been developed in Japan.

Over several years, many LC–MS and LC–MS/MS methods were reported for the simultaneous analysis of trichothecene mycotoxins, including DON and NIV. These methods have been applied to the hygienic control and surveillance of mycotoxins. However, LC–MS requires the use of expensive internal standards such as isotopically substituted compounds. Meanwhile, the precision of LC–UV has been valued, although its sensitivity is lower than that of LC–MS.

A selective analytical method based on HPLC, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for the simultaneous determination of NIV and DON. A liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI–MS) method based on time-of-flight MS (TOF/MS) with a real-time reference mass correction technique was also developed for the simultaneous determination of Fusarium mycotoxins (NIV, DON, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT-2 toxin, T-2 toxin, diacetoxyscirpenol, ZEN) and Aspergillus mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2) in corn, wheat, cornflakes, and biscuits [112].

Sulyok et al. [113] reported the first validated method for the determination of 39 mycotoxins in wheat and maize by liquid chromatography with electrospray ionization–triple quadrupole mass spectrometry (LC/ESI–MS/MS) without the need for any cleanup. The 39 analytes included A and B trichothecenes (including deoxynivalenol-3-glucoside), ZEN and related derivatives, fumonisins, enniatins, ergot alkaloids, ochratoxins, aflatoxins, and moniliformin, and six trichothecene mycotoxins (NIV, DON, fusarenon-X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin).

Taken together, these analytical methods should be chosen for any purpose. For surveillance, GC–MS and LC–MS/MS are efficient because of their sensitivity, and for enforcement of regulation, HPLC would be useful.

9 Exposure Assessment of NIV and Regulation

Exposure to mycotoxins depends on the contamination level in different foods and on the intake of those foods, which is influenced by the dietary culture of each country. These data are based on established regulation levels of individuals or international institutes. For mycotoxins, it is ideal to correct the actual monitoring data over several years in raw and processed food. Especially, since exposure to trichothecenes and their toxicity are of concern in young children, it is important to assess the intake by age layers. FAO/WHO organized the Global Environment Monitoring System/Food Contamination Monitoring and Assessment Programme (GEMS/Food) and utilizes this information to assess the intake of mycotoxins (Table 100.7).

Table 100.7 Analytical methods for determination of NIV
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9.1 Occurrence of NIV in Food and Feed

Compared to the worldwide distribution of DON, NIV contamination is found only in limited areas. In Fig. 100.5, the commodities contaminated with NIV that have been reported are plotted [120]. Contamination of cereals (wheat, oats, barley, maize, rice, rye) has been frequently found in Far East Asia (China, Korea, Japan), Southeast Asia (the Philippines, Vietnam), Oceania (New Zealand, Australia) Europe (Germany, Poland, Norway, the Netherlands), and Eastern Europe (Lithuania). The contamination of soybeans (50 μg/kg of NIV) has also been reported in Australia [121].

Fig. 100.5
figure 01355

Distribution of food contaminated with NIV (Modification cited by Ref. [120])

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From SCOOP data [11], food and food raw materials were shown to be contaminated by trichothecenes (DON, NIV, FX, T-2, and HT2 toxin, T-2 triol, diacetoxyscirpenol, neosolaniol, and verrucarol).

Table 100.8 shows NIV contamination in European countries. Among the cereals, mainly wheat, oats, wheat, barley, and rye are exposed to NIV. In Europe, the level and frequency of DON contamination are often higher than NIV contamination. Thus, it is generally believed that DON is more predominant than NIV and that their contamination levels shift in a parallel manner, i.e., when the DON level decreases, the NIV level also decreases. However, in the southern island of Japan (Kyushu), only the NIV-producing group strain exists (described in 1.3. mycology). Since commodities harvested from this area are contaminated with NIV, it has become a serious problem for human health.

Table 100.8 NIV contamination in food in European Union member states
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Far East countries, including Japan, Korea, and China, are also suffering from NIV contamination of grain. In Japan, NIV concentration in barley was higher than that in wheat, while in Korea, barley was the most susceptible commodity contaminated with NIV. NIV contamination in rice has been reported in Korea but not in Japan. Since rice is the dietary staple in most Far East countries, even if the contamination level is low, it is of concern to human health (Table 100.9).

Table 100.9 Occurrence of nivalenol in wheat in Eastern Asia (2002–2007)
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9.2 NIV Intake

The surveillance of NIV in food and estimated NIV intake were conducted in limited countries [22, 126]. Table 100.10 shows estimated NIV intake in the EU, UK, and Japan.

Table 100.10 Estimated NIV intake
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As shown in Table 100.10, viewing the entire population, the highest mean and 95th percentile of estimated NIV intake was shown in Austria. Among the exposed adults, in France, the estimated intake of NIV was highest compared to adults of other countries. However, the intake by children was higher than by adults. In young children, mean intake in the UK was 62–64 ng/kg bw/day, and these values were higher than in Japanese children.

9.3 International Evaluation

The FAO/WHO Joint Expert committee of Food Additives has not evaluated NIV contamination levels. The IARC has evaluated carcinogenesis of the toxins produced from F. graminearum, F. culmorum, and F. crookwellense, such as ZEN, DON, and acetyl NIV [127]. Their conclusion was that metabolites of these three Fusarium species should be placed in group 3, i.e., not classified as carcinogenic for humans.

The Scientific Committee for Food (EC) reported values for DON in 1999, for NIV in 2000, and for T-2 and HT-2 toxins in 2002[128]. According to their opinion, temporary tolerable daily intake (t-TDI) of NIV was estimated to be 0.7 μg/kg based on NOAEL and 0.7 mg/kg temporary bw based on the results of 1- and 2-year repeated dose studies reported by Ref. [77, 88].

In 2010, the FSC of Japan evaluated DON and NIV and concluded that the provisional maximum tolerable daily intake (PMTDI) of NIV was 0.4 μg/kg bw/day based on LOAEL (0.4 mg/kg bw/day) of subacute repeated dose toxicity studies using purified NIV [86]. As the PMTDI of DON was adopted to be 1.0 μg/kg bw, it had the same value as JECFA and EFSA.

9.4 Regulation

To date, no country has established a standard for NIV yet. However, regarding the risk assessment of NIV, Japan and EU lead the world. These countries have serious problems related to the management of NIV-producing fungi. The establishment of a regulation level for NIV is one of the effective means to prevent contamination of NIV in cereal grain.

10 Conclusion

Because NIV is a minor mycotoxin in the world, information for risk assessment is very poor. Of course, European countries and Japan have recognized the threat posed by NIV to human health and have started to evaluate this risk. In the risk assessment of NIV, it is recommended to take into account the effect of co-contamination with other trichothecene mycotoxins, namely, DON. As further studies, combination toxicology studies of NIV and DON are needed.