Mycotoxins are fungal secondary metabolites which, if ingested, can evoke a wide range of toxic responses and disease conditions in higher vertebrates. Cyclopiazonic acid (α-cyclopiazonic acid; CPA, Figure 1) is an indole-tetramic acid mycotoxin produced by the ubiquitous genera of molds Aspergillus and Penicillium. Beside colonizing various grains and seeds [1,2], these molds can grow on many food substrates, such as cheese and meat products [3,4,5]. Therefore, CPA can contaminate a number of agricultural commodities, animal feeds, and food sources. This toxin has been found in edible tissue in poultry, milk, and eggs [6,7,8] presumptively due to animals’ consumption of contaminated feeds. Despite the wide presence of CPA, few incidents of animal mycotoxicosis and no confirmed incident of human poisoning have been attributed to CPA. Despite its early discovery, the benign nature of CPA has rendered it to receive much less attention of the mycotoxin research community than its counterparts such as aflatoxins, trichothecenes, fumonisins and ochratoxins in the past two decades.

The chemistry and biochemistry of the synthesis of CPA, which was carried out primarily in a Penicillium strain, received considerable attention in the 1970s. Some intermediates and enzymes involved in their formation and/or conversions were purified (for a review, see [9] and references therein). The chemical characterization of CPA also has elicited a tremendous interest in studies of biologically active natural products containing the tetramic acid structural motif, an important class of nitrogen-containing heterocycles [10,11,12]. The detailed revelation of CPA formation, especially the aspects of molecular biology and enzymatic mechanisms, however, had been lacking until the recent identification of three clustered biosynthetic genes in Aspergillus flavus and closely related Aspergillus oryzae [13,14,15]. This review aims to provide an historic overview of CPA studies and the most recent progress made in the elucidation of CPA biosynthesis.

Cyclopiazonic acid is named after the strain, Penicillium cyclopium Westling [16], from which it was originally isolated. However, P. cyclopium or its synonym P. aurantiogriseum [17] have not been found to make CPA, and the CPA-producing strain originally isolated (CSIR 1082) was later identified as P. griseofulvum Dierckx [18]. Other species of Penicillium including P. griseofulvum, P. camemberti, P. urticae and P. commune have been reported to consistently produce CPA [19]. Other species of P. chrysogenum, P. nalgiovense,P. crustosum, P. hirsutum and P. viridicatum also have been reported to produce CPA [20] but CPA production by these taxa has not been confirmed. Certain Aspergillus species such as A. flavus, A. oryzae, A. fumigatus, A. versicolor, and A. tamarii also produce CPA [21,22,23]. In a recent survey, Vinokurova et al. [24] found that 30% of the A. fumigatus and A. phoenicis strains but only one of 21 A. versicolor strains were able to produce CPA. The low incidence of CPA production by A. versicolor warrants a further examination since A. versicolor Tiraboschi originally reported to produce CPA [25] was later identified as A. oryzae [26].

CPA and aflatoxins often co-contaminate crops [41,42,44,45,46,47,48]. The source of these mycotoxins is complicated by the fact that both aspergilli and penicillia are often found in the same crop and as well as after grain storage [49,50,51,52,53]. The most important aflatoxin producers are members in the Aspergillus section Flavi, particularly A. flavus, A. parasiticus, and A. nomius. Some strains of other members such as A. pseudotamarii [54], A. tamarii, A. toxicarius, and A. bombycis [55] also have been reported to produce aflatoxins. A. flavus has been divided into two subtypes based on sclerotial size [56,57,58]. The type with small sclerotia (S_B or A. flavus Group IB) is found together with the L-morphotype (large sclerotia > 400 um) as crop and soil contaminants on all continents except Antarctica. Small sclerotia-producing isolates from crops in Thailand that resemble A. flavus S_B strains but produce both B- and G- aflatoxins have been called A. flavus var. parvisclerotigenus [59]. This taxonomic identification has never been recognized as authoritative, and in an independent investigation of Aspergillus section Flavi isolates from Thailand certain isolates with small sclerotia and the ability to produce both B- and G- aflatoxins have been identified as a variant clade of A. nomius rather than A. flavus [60]. Atypical A. flavus isolates (also called strain S_BG or A. flavus Group II), which produce small sclerotia and are intermediate between A. flavus and A. parasiticus have been identified in soil and agricultural samples from West Africa, Argentina and Australia [28,58,61]. These S_BG isolates have recently been classified as A. minisclerotigenes and A. arachidicola [62]. Only A. minisclerotigenes produces CPA. Isolates of these new taxa are also possibly found in the United States (Texas) [58,61] and Europe (Italy, Portugal, and Spain) [37,38,40]. Since the original finding of CPA production by A. flavus [63], the relationship between CPA and aflatoxin production by A. flavus and the closely related A. parasiticus has been investigated. Typical A. flavus isolates can produce only B-type aflatoxins and CPA, and A. parasiticus always produces B- and G-type aflatoxins but never CPA [23]. Table 1 summarizes the ability of A. flavus isolates to produce CPA and aflatoxins from various regions of the world. The atypical B- and G-type producers are excluded in Table 1. As can be seen, the ability of A. flavus isolates to produce CPA varies greatly. The incidence of CPA production by A. flavus is high (>70%) in Argentina and in the United States (Table 1).

The toxicity of CPA is attributed to its ability to alter normal intracellular calcium flux through the specific inhibition of sarcoplasmic or endoplasmic reticulum calcium-dependent ATPase (SERCA) essential for calcium uptake as in the muscle contraction-relaxation cycle, which results in increased muscle contraction [64,65]. SERCA also plays a crucial role in other body cells. It serves a housekeeping function, maintaining high calcium in the endoplasmic reticulum and low cytosolic calcium. This calcium gradient is vital for the cell and controls proliferation, differentiation and cell death. Most recent evidence shows that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of four transmembrane helices of the ATPase [66]. Besides the specific inhibition of ATPase activity, CPA induced various pathological lesions in test animals [19].

In rats, intraperitoneal injection of CPA has been reported to cause lesions of the liver, kidney, pancreas, spleen, and heart [67]. The oral LD₅₀ value for CPA in nonpregnant mice was 64 mg/kg body weight, and the toxicity syndromes included ptosis, hypokinesia, hypothermia, tremor, cessation of food and water intake and resulting cachexia. The intensity of these toxic syndromes was dose-dependent. In pregnant mice, a single oral pulse dose of CPA (15 to 50 mg/kg) decreased body weight gain and the pregnancy rates significantly [68].In feeding studies broiler chickens fed 100 ppm of CPA had a high mortality rate and decreased weight gain. Postmortem examination showed that the chickens had proventricular lesions characterized by mucosal erosion and hyperemia, yellow foci in livers and spleens. Microscopic examination of tissues showed ulcerative proventriculitis, mucosal necrosis in the gizzard, and hepatic and splenic necrosis and inflammation. Chickens fed 50 ppm CPA had thick mucosa and dilated proventricular lumens as well as hyperplasia of the proventricular mucosal epithelium. CPA at 10 ppm, however, did not cause significant lesions [22]. Dogs administered CPA at 0.5 mg/kg or 1.0 mg/kg died before the end of 90-day study period, which suggests a much lower no-observable-effect level (NOEL) than other small animals. Gross lesions of diffuse hyperemia with focal areas of hemorrhage and ulceration and microscopic lesions of necrosis, vasculitis, lymphoid necrosis, and karyomegaly in organs were apparent [69]. Surprisingly, among the large mammals, pigs appear to be quite sensitive to CPA with a NOEL of approximately 1.0 mg/kg [70].

The effects of CPA (50 mg/kg) and aflatoxin (3.5 mg/kg) on broiler chickens, in most cases, were additive [71]. Chickens treated with CPA and aflatoxin had increased weights of the liver, kidney, and pancreas, showed signs of proventriculitis, had decreased concentrations of serum albumin and phosphorus, and increased concentrations of blood urea nitrogen and serum glutamic oxalacetic transaminase. Postmortem examination showed that the chickens fed CPA and aflatoxin exhibited similar pathological effects as those fed CPA alone, which included thickened mucosa and dilated proventricular lumens, hard fibrotic spleen, and atrophy of the gizzard.

The IC₅₀ of CPA on cell proliferation was 40-fold lower than ochratoxin (OTA) in an in vitro test using purified lymphocytes from piglets [72]. The combined effect of CPA and OTA based on IC₂₀ on the proliferation of porcine lymphocytes was additive [73]. The individual and combined effect of CPA (34 mg/kg) and OTA (2.5 mg/kg) to broiler chickens were examined [74]. CPA reduced body weight to about 80% of the control, but it did not affect the relative weight of liver, kidney, pancreas except for proventriculus. The interaction of CPA and OTA was additive. The pathological lesions were similar to those caused by CPA and aflatoxin. Animals had increased weights of liver, kidney, pancreas, and proventriculus, decreased serum total protein and albumin, and increased triglycerides, uric acid and creatine kinase activity; creatine is important in maintaining the ATP level for skeletal muscle contraction. Postmortem examination revealed similar pathological lesions as those caused by CPA and aflatoxin.

The feeding of CPA (34 mg/kg) and T-2 toxin (6 mg/kg) also caused reduced performance and adversely affected the health of broiler chickens [75]. A synergistic interaction between CPA and T-2 for relative liver and kidney weights, serum cholesterol and triglyceride concentrations was observed. This combination also affected the 3-week body weight significantly. CPA and T-2 can induce cell death in lymphoid organs [76]. The immunopathological effect of 10 ppm CPA and 1 ppm T-2 in combination included decreases in both thymic CD₈⁺ lymphocyte and splenic CD₄⁺ and CD₈⁺ lymphocytes [77].

CPA is not a potent acute toxin because its oral LD₅₀ in rats is in the range of 30 to 70 mg/kg [68,78]. Few incidents of animal mycotoxicosis have been reported because of the benign nature of CPA, the small amounts usually present, or the fact its effects may be masked by other concurrent mycotoxins. Both CPA and aflatoxins were isolated from peanut meal related to the turkey ‘X’ disease that caused the death of 100,000 turkeys in England in the early 1960s [79,80]. Although aflatoxins were regarded as the main culprit, CPA likely contributed to some of the observed pathological clinical signs, such as catarrhal or haemorrhagic enteritis and opisthotonus that are not characteristic of aflatoxicosis [79,81].

Despite the CPA-induced pathological effects in test animals, field incidents of CPA mycotoxicoses in humans have not been reported. Nonetheless, it has been speculated that CPA may be associated with ‘kodo poisoning,’ a toxic reaction characterized by nausea, vomiting, depression, intoxication and unconsciousness after consumption of CPA-contaminated Kodo millet in some parts of North India [78,82]. Based on a NOEL of 1 mg/kg/day and taking into consideration species variability, for humans approximately 10 μg/kg/day or 700 μg/day was suggested as the maximum acceptable daily intake (ADI) dose [19].

Chemically, CPA belongs to the family of indole-tetramic acid secondary metabolites. P. griseofulvum Dierckx (originally called P. cyclopium) showed the maximal rate of CPA production after cessation of growth in Czapek’s medium. Research to elucidate the mechanism of CPA biosynthesis was carried out primarily in this strain in the 1970s. The origin of the carbon skeleton of CPA was established by feeding studies using radiolabeled substrates [83]. Chemical degradation analysis indicated that CPA is derived from tryptophan, a C5-unit formed from mevalonic acid and two molecules of acetic acid. The incorporation efficiencies of sodium [1-¹⁴C]acetate, [2-¹⁴C] mevalonic acid, and indole-labeled tryptophan were 3.5%, 7.0%, and 24.7%, respectively. The efficient incorporation of labeled tryptophan strongly suggested that it is a direct precursor of CPA. Further studies showed that all carbon atoms of the tryptophan were incorporated into CPA [84]. The presence of both β-CPA (bis-secodehydrocyclopiazonic acid) and CPA in mycelia suggested that β-CPA may be a precursor [85]. As further evidence for β-CPA being the precursor, the time-course of metabolite production showed that, when only trace amounts of CPA were produced, β-CPA predominated, while as CPA formation increased, the amount of β-CPA declined. After feeding [1-¹⁴C]acetate-labeled β-CPA 67% was converted to CPA, while 18% remained as β-CPA [83]. The compound γ,γ-dimethylallyltryptophan was known to be a precursor of ergot alkaloids [86,87,88]. Incubation of a cell-free-extract (CFE) with a mixture of radiolabeled [³H]tryptophan and [1-¹⁴C] dimethylallyl diphosphate (DMAPP) did not yield CPA labeled with either ³H or ¹⁴C although the resulting β-CPA contained 19% of the added ¹⁴C, but no ³H [89]. This result showed that the labeled DMAPP, but not tryptophan could be incorporated into β-CPA by the CFE. This study showed that CPA formation was different from ergot alkaloid biosynthesis, in the latter biosynthesis involves direct prenylation of tryptophan by DMAPP [86,90]. Subsequent studies found that the substrate for DMAPP in the CFE was α-acetyl-γ-(β-indolyl)methyltetramic acid (cycloacetoacetyl-L-tryptophan, cAATrp) [84]. The CFE converted exogenously supplied cAATrp and DMAPP into β-CPA in 1:1 stoichiometry. The responsible enzyme in the CFE was designated as cycloacetoacetyltyptophanyl dimethylallyl transferase. This enzyme, also called β-CPA synthase or dimethylallyl cycloacetoacetyl typtophan synthase (DCAT-S) was purified by McGrath et al. (1977). It has a molecular weight of 95 kDa with an isoelectric point of 5.3. Its concentration reached a maximum at 60 hr during fermentation. This synthase is very specific for DMAPP and cAATrp and could not use isopentenyl diphosphate (IPP, an isomer of DMAPP), tryptophan or N-acetyltryptophan as substrates [91]. The dimethylallyl tryptophan synthases of Clavices purpurea, Aspergillus fumigatus, Aspergillus nidulans and Malbranchea aurantiaca for alkaloid biosynthesis are α₂ dimers [86,92] suggesting that the β-CPA synthase is also likely to be a dimer. The enzyme(s) for the formation of cAATrp was never isolated from P. griseofulvum Dierckx. Schabort et al. [93] isolated five isozymes, designated as β-cyclopiazonate oxidocyclase that catalyzed oxidation and cyclization of β-CPA to CPA; each of the β-cyclopiazonate oxidocylases has a covalently linked flavin. Spectrophotometric titration studies of oxidized β-cyclopiazonate oxidocyclase with substrate analogs showed that the NH group in the tetramic acid residue is involved in binding to the active site [94,95].

The gene preceding maoA of the CPA gene cluster in A. oryzae RIB40 and NBRC 4177 encodes a P450 monooxygenase while in the same relative location in A. flavus NRRL3357 and AF13 no such a gene is present (Figure 3). The unique location of this p450 gene (=cpaC) in A. oryzae is reminiscent of the same gene organization in the Aspergillus S_BG isolate BN008R (ATCC MYA379) collected from Benin, West Africa [61,121]. A comparison of the 12 kb region beyond the norB gene in BN008R to the corresponding region in RIB40 shows that they share > 95% nucleotide identity (>98% in the maoA and the p450 coding regions). A. oryzae mutants with p450 deleted failed to produce mono- and dihydroxy CPA when compared to the wild type [122]. Since the p450 gene is missing in CPA-producing A. flavus NRRL3357 and AF13 (Figure 3), it is not directly involved in CPA biosynthesis. It is not known whether A. oryzae RIB40 gained the p450 gene or A. flavus NRRL3357 lost the p450 gene.

RIB40 and NRRL3357 exhibit other unique differences; the former has the type I deletion in its norB-cypA region, an S-strain signature, while the latter has the type II deletion that is often associated with aflatoxigenic L-strain A. flavus isolates [123]. They also have distinct single nucleotide polymorphisms in the omtA and dmaT genes involved in aflatoxin and CPA biosynthesis, respectively [58,123] (and unpublished results). A. flavus populations are genetically diverse. A. oryzae is believed to be selected or to have evolved from certain groups of nonaflatoxigenic A. flavus. A. oryzae strains are classified into three groups based on deletion patterns in the aflatoxin gene cluster [124,125]. Group 1 has all aflatoxin gene orthologs, group 2 has the region beyond the ver1 gene deleted but contains a DNA segment of unknown origin, and group 3 has the partial aflatoxin gene cluster up to the vbs gene (see Figure 3). Group 3 resembles the E, F, and G deletion patterns in nonaflatoxigenic and CPA-negative A. flavus isolates [101], which lack the genome region beyond the respective partial aflatoxin gene cluster (connected to telomeric repeat). Since the CPA gene cluster in A. oryzae resides next to the aflatoxin gene cluster as in A. flavus, group 3 likely has lost the CPA gene cluster. Group 2 strain RIB62 has been confirmed to be lacking the CPA gene cluster resulting from a large deletion [125]. Some strains of group 1 A. oryzae still maintain the ability to produce CPA, such as NBRC 4177 while other strains have lost the ability, such as RIB40 due to a truncation that caused the loss of more than half of the pks-nrps gene.

A PCR survey of the strains of A. oryzae and A. flavus and related A. parasiticus and A. sojae (Table 3) showed that only isolates of nonaflatoxigenic and CPA-positive L-strain A. flavus, with the type I, S-strain genetic signature in a previously characterized clade, for example, MS1-1, NC3-6, SC3-5 and TX21-9 [123] also have the p450 gene.

As mentioned earlier, atypical A. flavus S_BG isolates have been classified into the new taxa of A. minisclerotigenes and A. arachidicola [62]. A. minisclerotigenes differs from A. arachidicola. in that it produces CPA like A. parvisclerotigenus, previously named A. flavus var. parvisclerotigenus by Saito and Tsuruta [59], which included largely S_B strains and only a small number of S_BG strains [56,59]. Genetic evidence shows that the β-tubulin gene sequence of the A. parvisclerotigenus strain, CBS 121.62^T, isolated from Nigeria by Hesseltine et al. [56], is different from those of the A. minisclerotigenes strains and this strain produced other metabolites, such as A30461 and speradine A not produced by A. minisclerotigenes [62].

Although Saito and Tsuruta [59], in their naming of A. flavus var. parvisclerotigenus, included morphologically similar S_BG isolates obtained by Hesseltine et al. [56], Ehrlich et al. [60] considered the S_BG isolates collected from different regions of Thailand by Saito and Tsuruta [59] a variant clade of A. nomius rather than A. flavus. The S_BGAspergillus BN008R isolated from Benin, West Africa [61] likely is A. minisclerotigenes. We hypothesize that the common ancestor of the aforementioned nonaflatoxigenic and CPA-positive L-strain A. flavus isolates and the group 1 A. oryzae that contain the p450 gene may be A. minisclerotigenes or A. flavus var. parvisclerotigenus. The loss of both B- and G-type aflatoxins renders them to be regarded as A. flavus due to the morphological and cultural similarities to other clades of A. flavus. Probably, two genetic variants are responsible for CPA production in A. flavus. One is the S_BA. flavus strains that always have the type I norB-cypA deletion, and another is L-strain A. flavus strains with the type II deletion, which are either aflatoxigenic or nonaflatoxigenic.

Like aflatoxins, the benefit of CPA to the producing fungi is not clear. CPA is an excellent chelator of iron. CPA production is greatest during the period preceding dormancy when growth has practically stopped. Because concentrations of free iron in soil are usually low, it would be beneficial to fungi to have a readily available supply of iron for subsequent growth. Riley and Goeger [126] speculated that stockpiles of CPA-chelated iron would be conducive to rapid fungal growth for occupying a niche in the saprophytic environments. Similar to a siderophore, which is an iron-chelating compound secreted by bacteria, fungi, and plants, CPA probably fulfilled partly this ion-chelating function long ago before a true siderophore took its place. Many siderophores are nonribosomal peptides and dissolve ions by chelation as soluble Fe³⁺ complexes that are taken up by active transport mechanisms. In the A. flavus genome, in the same subtelomeric region where the CPA gene cluster resides, sidC and sidT (msf2) genes encoding a siderophore and a siderochrome-iron transporter, respectively, have been identified [13]. Their location, which is at a terminus of chromosome 3, suggests that they were acquired later than the CPA gene cluster to carry out the iron-chelating function.

The CPA and aflatoxin gene clusters are contiguous on the subtelomeric region of chromosome 3 in the A. flavus and A. oryzae genomes. The enzyme functions of the three CPA genes correlate well with current as well as previous studies of the biosynthesis of this indole-tetramic acid product. Although CPA is not a potent acute toxin and few incidents of mycotoxicoses have been reported, aflatoxin B₁ is a potent carcinogen. Little is known about the synergistic effects of the two toxins and more research is needed especially because plans are being considered to apply nonaflatoxigenic A. flavus strains in large amounts to control aflatoxin contamination in corn, cottonseed, and peanuts and some A. oryzae strains used in food fermentation are capable of CPA production. With knowledge of the co-localization of the two gene clusters it is now easy to explain why strains of A. flavus and A. oryzae have different abilities to produce aflatoxin and CPA. This understanding has significant health implications. The genetic diversity of A. flavus and A. oryzae in the region adjoining the CPA gene cluster suggests a divergence of A. flavus from A. oryzae. We suggest that A. oryzae most likely descended from an ancestor that was the precursor of the Aspergillus S_BG variant, while A.flavus descended from a precursor of A. parasiticus.