Timothy D. Phillips

OXIDATIVE DEGRADATION AND DETOXIFICATION OF MYCOTOXINS USING A NOVEL SOURCE OF OZONE

Practical methods to degrade mycotoxins using ozone gas (O3) have been limited due to low O3 production capabilities of conventional systems and their associated costs. Recent advances in electrochemistry (i.e. proton-exchange membrane and electrolysis technologies) have made available a novel and continuous source of O3 gas up to 20% by weight. It is possible that the rapid delivery of high concentrations of O3 will result in mycotoxin degradation in contaminated grains--with minimal destruction of nutrients. The major objectives of this study were to investigate the degradation and detoxification of common mycotoxins in the presence of high concentrations of O3. In this study, aqueous equimolar (32 microM) solutions of aflatoxins B1 (AfB1), B2 (AfB2), G1 (AfG1), G2 (AfG2), cyclopiazonic acid (CPA), fumonisin B1 (FB1), ochratoxin A (OA), patulin, secalonic acid D (SAD) and zearalenone (ZEN) were treated with 2, 10 and/or 20 weight% O3 over a period of 5.0 min and analysed by HPLC. Results indicated that AfB1 and AfG1 were rapidly degraded using 2% O3, while AfB2 and AfG2 were more resistant to oxidation and required higher levels of O3 (20%) for rapid degradation. In other studies, patulin, CPA, OA, SAD and ZEN were degraded at 15 sec, with no by-products detectable by HPLC. Additionally, the toxicity of these compounds (measured by a mycotoxin-sensitive bioassay) was significantly decreased following treatment with O3 for 15 sec. In another study, FB1 (following reaction with O3) was rapidly degraded at 15 sec, with the formation of new products. One of these appeared to be a 3-keto derivative of FB1. Importantly, degradation of FB1 did not correlate with detoxification, since FB1 solutions treated with O3 were still positive in two bioassay systems.

pH Regulation of Sterigmatocystin and Aflatoxin Biosynthesis in Aspergillus spp.

ABSTRACT Aflatoxin (AF) and sterigmatocystin (ST) are toxic secondary metabolites produced by the same biochemical pathway found in several Aspergillus spp. The expression of the homologous ST/AF structural gene, stcU in A. nidulans and ver-1 in A. parasiticus, was affected by external pH of liquid growth media. Both stcU and ver-1 mRNAs appeared earlier and were expressed at higher levels in cultures grown in acidic media (pH 4 to 6) versus neutral (pH 7) and alkali (pH 8) media. Transcript levels correlated with ST/AF production. Visual and spectrophotometric analysis of production of the orange ST/AF intermediate, norsolorinic acid (NOR), also paralleled transcript patterns and indicated that the pH effects were operative in different nitrogen- and carbon-based solid growth media. Five- to 10-fold increases in ST, AF, and NOR were measured in cultures grown in pH 4 or 5 versus pH 8 media. An A. nidulans strain carrying a mutation resulting in constitutive activity of the pH regulatory factor, PacC, produced 10-fold less ST than did wild type. The stcU transcript was not noticeably affected by pH in this strain. The results suggest a general pattern of pH regulation of ST/AF biosynthesis that may override previously noted carbon and nitrogen effects.

Protective grass endophytes : Where are they from and where are they going ?

erhaps the most widely used agents of biological plant protection are endophytic fungal symbionts (endophytes) of forage and turfgrasses. These are fungi of the family Clavicipitaceae, which grow between host cells in vegetative tissues, ovules, and seeds of systemically infected grass plants. The existence of these endophytes was not fully appreciated until recent years, although the protection they provide against insect damage (Fig. 1) and drought contributes to the superior agronomic qualities of favorite pasture grasses in North America, Australia, and New Zealand. Unfortunately for livestock farmers, these endophytes also provide a degree of protection from grazing mammals. In 1977, Bacon et al. (2) reported that the grass Festuca arundinacea var. genuina Schreb. (hexaploid tall fescue) had a fungal endophyte related to Epichloe typhina (Pers.:Fr.) Tul., and that this endophyte— now known as Neotyphodium coenophialum (Morgan-Jones & Gams) Glenn, Bacon, & Hanlin—was responsible for toxicosis suffered by livestock grazing the grass. Epichloe species were known for many decades (44), but reports relating that nonpathogenic endophytes could be detrimental to livestock provided new impetus for intensive studies, making the grass–endophyte associations among the best characterized symbioses in biology. Less than two decades of research have yielded a rich body of knowledge about these symbioses: their secondary product chemistry, ecology, evolution, genetics, and molecular biology; their ecological roles as protectants from insect and vertebrate herbivores, pathogenic fungi and nematodes, and drought; and their effects on host growth and competitiveness. The endophytes produce numerous alkaloids, some of which are unrelated to any known from plants or other fungi. Their genetic and evolutionary complexity is extraordinary. They were the first fungi genetically documented as interspecific hybrids (47,53). Meanwhile, molecular genetic techniques were applied to the endophytes of tall fescue and other grasses, bringing us closer to the prospect of reducing or eliminating their toxicosis to livestock while continuing to employ their bioprotective qualities.

Reducing human exposure to aflatoxin through the use of clay: a review.

Innovative sorption strategies for the detoxification of aflatoxins have been developed. NovaSil clay (NS) has been shown to prevent aflatoxicosis in a variety of animals when included in their diet. Results have shown that NS clay binds aflatoxins with high affinity and high capacity in the gastrointestinal tract, resulting in a notable reduction in the bioavailability of these toxins without interfering with the utilization of vitamins and other micronutrients. This strategy is being evaluated as a potential remedy for acute aflatoxicosis, and as a sustainable human intervention for aflatoxins via the diet. Phase I and II clinical trials confirmed the apparent safety of NS for further study in humans. A recent study in Ghanaians at high risk for aflatoxicosis has indicated that NS (at a dose level of 0.25%) is effective in decreasing biomarkers of aflatoxin exposure and does not interfere with the levels of serum vitamins A and E, and iron and zinc. In summary, enterosorption strategies/therapies based on NS clay are promising for the management of aflatoxins and as a sustainable public health intervention. The NS clay remedy is novel, inexpensive and easily disseminated. Based on the present research, aflatoxin sequestering clays should be rigorously evaluated in vitro and in vivo, and should meet the following criteria: (1) favourable thermodynamic characteristics of mycotoxin sorption, (2) tolerable levels of priority metals, dioxins/furans and other hazardous contaminants, (3) safety and efficacy in multiple animal species, (4) safety and efficacy in long-term studies, and (5) negligible interactions with vitamins, iron and zinc and other micronutrients.

G-protein signalling mediates differential production of toxic secondary metabolites.

Filamentous fungi elaborate a complex array of secondary metabolites, including antibiotics and mycotoxins. As many of these compounds pose significant economic and health concerns, elucidation of the underlying cellular mechanisms that control their production is essential. Previous work revealed that synthesis of the carcinogenic mycotoxins sterigmatocystin (ST) and aflatoxin (AF) in Aspergillus species is negatively controlled by FadA, the α‐subunit of a heterotrimeric G‐protein. In sharp contrast, we show here that the dominant activating fadA allele, fadAG42R, stimulates transcription of a gene from the A. nidulans penicillin (PN) gene cluster and elevates penicillin production. Thus, FadA has opposite roles in regulating the biosynthesis of a potent antibiotic (PN) and a lethal mycotoxin (ST) in A. nidulans. Furthermore, expression of fadAG42R in Fusarium sporotrichioides increases trichothecene (TR) mycotoxin production and alters TR gene expression. Our findings reveal that a G‐protein defines an important control point for differential expression of fungal secondary metabolites within and across fungal genera. These data provide critical evidence suggesting that targeting G‐protein signal transduction pathways as a means of controlling or preventing the production of a single mycotoxin could have serious undesirable consequences with regard to the production of other secondary metabolites.

Adsorption of Salmonella enteritidis by cetylpyridinium-exchanged montmorillonite clays.

Recent experiments in our laboratory have suggested that certain montmorillonite clays, when exchanged with the cationic surfactant cetylpyridinium (CP), may be useful in removing bacteria from aqueous solution. During an initial study, screening various CP-exchanged products for potential antibacterial activity, three CP-exchanged clays - CP*AAM (acid-activated montmorillonite), CP*STx-1 (Ca(++)-montmorillonite), and CP*SWy-2 (Na(+)-montmorillonite), proved to be the most effective. Binding studies were performed using 1mg each of CP-exchanged AAM, STx-1, and SWy-2 with a standardized Salmonella enteritidis solution containing approximately 40,000 colony forming units (CFU)/ml. The modified clays reduced bacterial numbers 98.1, 97.6, and 95.2%, respectively. In contrast, the parent clays only produced reductions of 39.8, 16.9, and 16.6%, respectively. Attempts were made to desorb CP from the modified clays by washing in sterile physiological saline for 24h. The resulting wash solutions failed to produce any significant reduction in bacterial colony counts; while, the washed clays retained their full antimicrobial activity. These findings suggested that the antibacterial effect of the clays is localized on the clay surface and is not due to CP dissociating from the clay. Electron microscopy revealed that the bacteria adhered to the surface of the CP-exchanged clays, but not the parent clays. Results from timed binding studies showed that the antibacterial effect was stable over the period observed. Rates of binding were positively influenced by increasing temperature, not affected by changes in pH, and negatively influenced by the presence of organic contaminants. The mechanism by which bacterial counts are reduced may involve the enhanced hydrophobicity and affinity of the CP-exchanged clay for Salmonella and the antibacterial activity of CP.

Aroclor 1254 as an antagonist of the teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 20 micrograms/kg) to pregnant C57BL/6J mice (on day 10) resulted in 62% fetuses with cleft palate per litter without any observable maternal toxicity. In contrast, Aroclor 1254 administered at a dose of 750 mumol/kg was not teratogenic. Cotreatment of the pregnant mice with both Aroclor 1254 (244 mg/kg) and 2,3,7,8-TCDD (20 micrograms/kg) resulted in an 8.2% incidence of cleft palate per litter. In contrast, Aroclor 1254 did not afford any protection from the teratogenicity of dexamethasone in C57BL/6J mice. Previous studies have shown that Aroclor 1254 can act as a partial antagonist of the microsomal enzyme induction and immunotoxic effects of 2,3,7,8-TCDD in C57BL/6J mice and this paper demonstrates that the commercial polychlorinated biphenyl mixture also antagonizes 2,3,7,8-TCDD-mediated teratogenicity in this strain of mice.

Characterization of Clay-Based Enterosorbents for the Prevention of Aflatoxicosis

Appropriate chemical interventions that can block, retard, or significantly diminish foodborne exposure to aflatoxins are high priorities. A practical and effective approach to the aflatoxin problem has been the dietary inclusion of a processed calcium montmorillonite clay (HSCAS). HSCAS acts as an enterosorbent that rapidly and preferentially binds aflatoxins in the gastrointestinal tract resulting in decreased aflatoxin uptake and bioavailability. In mechanistic studies, we have shown that the intact dicarbonyl system in aflatoxin is essential for optimal sorption by HSCAS. Evidence also suggests that aflatoxins react at multiple sites on HSCAS clay surfaces (especially those within the interlayer region). Due to conceivable risks associated with the dietary inclusion of nonspecific binding agents, all aflatoxin enterosorbents should be tested in sensitive animal models for efficacy, safety, and the potential for nutrient interactions.

A Novel Regulatory Gene, Tri10, Controls Trichothecene Toxin Production and Gene Expression

ABSTRACT We report here the characterization of Tri10, a novel regulatory gene within the trichothecene gene cluster. Comparison of Tri10 genomic and mRNA sequences revealed that removal of a single 77-bp intron provided a 1,260-bp open reading frame, encoding a 420-amino-acid protein. Disruption ofTri10 in Fusarium sporotrichioidesabolished T-2 toxin production and dramatically decreased the transcript accumulation for four trichothecene genes (Tri4, Tri5, Tri6, and Tri101) and an apparent farnesyl pyrophosphate synthetase (Fpps) gene. Conversely, homologous integration of a disruption vector by a single upstream crossover event significantly increased T-2 toxin production and elevated the transcript accumulation of the trichothecene genes andFpps. Further analysis revealed that disruption ofTri10, and to a greater extent the disruption ofTri6, increased sensitivity to T-2 toxin under certain growth conditions. Although Tri10 is conserved inFusarium graminearum and Fusarium sambucinum and clearly plays a central role in regulating trichothecene gene expression, it does not show any significant matches to proteins of known or predicted function or to motifs except a single transmembrane domain. We suggest a model in which Tri10acts upstream of the cluster-encoded transcription factor TRI6 and is necessary for full expression of both the other trichothecene genes and the genes for the primary metabolic pathway that precedes the trichothecene biosynthetic pathway, as well as for wild-type levels of trichothecene self-protection. We further suggest the presence of a regulatory loop where Tri6 is not required for the transcription of Tri10 but is required to limit the expression of Tri10.

Efficacy of Zeolitic Ore Compounds on the Toxicity of Aflatoxin to Growing Broiler Chickens

Commercially available zeolitic ore compounds, when incorporated into the diets at 0.5%, were evaluated for their ability to reduce the deleterious effects of 3.5 mg aflatoxin/kg feed on growing broiler chickens from 1 day to 3 weeks of age. In a series of four experiments, the compounds used included the following: mordenite (particle size of -20 mesh; Zeomite); clinoptilolite (particle size of -20 mesh; Zeobrite); SC Zeolite (particle size of -20 mesh); and clinoptilolite (particle size of -35 mesh; Clino 1) or clinoptilolite (particle sizes of -20 plus +35 mesh; Clino 2). Results demonstrated that 0.5% Zeobrite, Clino 1, or Clino 2 added to aflatoxin-contaminated diets did not significantly (P < 0.05) diminish the toxicity of high concentrations of aflatoxin to growing broiler chicks. Zeomite mordenite ore reduced the toxicity of aflatoxin to growing chicks by 41%, as indicated by weight gains, liver weight, and serum biochemical measurements, which compares favorably with its in vitro binding capacity to aflatoxin. SC Zeolite reduced weight-gain toxicity of aflatoxin by approximately 29%.