Hamed K. Abbas

An overview of mycotoxin contamination in foods and its implications for human health

Mycotoxins are natural contaminants of cereals and other food commodities throughout the world and they significantly impact human and animal health. Mycotoxins are toxic secondary metabolites produced by species of filamentous fungi growing on grains before harvest and in storage. When ingested, inhaled, or absorbed through skin, mycotoxins may reduce appetite and general performance, and cause sickness or death in humans. Mycotoxins subject to government regulation in most countries include aflatoxins, fumonisins, ochratoxins, deoxynivalenol, zearalenone, and patulin, produced by species of Aspergillus, Fusarium, and Penicillium. Aflatoxins, fumonisins, and ochratoxins pose the most serious threats to human health worldwide. This review describes the prevalence of mycotoxins in foods and its implications on human health, which may help in establishing and carrying out proper management strategies. Data from detailed investigations of food mycotoxins worldwide help provide safer food for consumption and help prioritize future research programs.

Evidence for disruption of sphingolipid metabolism as a contributing factor in the toxicity and carcinogenicity of fumonisins

Fumonisins are inhibitors of the biosynthesis of sphingosine and more complex sphingolipids. In eucaryotic cells, fumonisin inhibition of sphingolipid biosynthesis is a result of inhibition of the enzyme ceramide synthase. Large increase in free sphinganine concentration in plant and animal cells are observed within a few hours after exposure to fumonisins and/or Alternaria toxins (AAL-toxins). Some of the sphinganine is metabolized to other bioactive intermediates, and some is released from cells. In animals, free sphinganine accumulates in tissues and quickly appears in blood and urine. Free sphingoid bases are toxic to most cells, and complex sphingolipids are essential for normal cell growth. Fumonisin B1 stimulates sphinganine-dependent DNA synthesis in Swiss 3T3 cells, but is mitoinhibitory in other cell types. In cultured cells the accumulation of bioactive long-chain sphingoid bases and depletion of complex sphingolipids are clearly contributing factors in growth inhibition, increased cell death, and (in Swiss 3T3 cells) mitogenicity of fumonisins. While disruption of sphingolipid metabolism directly affects cells, it may indirectly affect some tissues. For example, fumonisin B1 impairs the barrier function of endothelial cells in vitro. Adverse effects on endothelial cells could indirectly contribute to the neurotoxicity and pulmonary edema caused by fumonisins. It is hypothesized that fumonisin-induced changes in the sphingolipid composition of target tissues could directly or indirectly contribute to all Fusarium moniliforme-associated diseases.

Biocontrol of aflatoxin in corn by inoculation with non-aflatoxigenic Aspergillus flavus isolates

Abstract The ability of two non-aflatoxigenic Aspergillus flavus Link isolates (CT3 and K49) to reduce aflatoxin contamination of corn was assessed in a 4-year field study (2001–2004). Soil was treated with six wheat inoculant treatments: aflatoxigenic isolate F3W4; two non-aflatoxigenic isolates (CT3 and K49); two mixtures of CT3 or K49 with F3W4; and an autoclaved wheat control, applied at 20 kg ha−1. In 2001, inoculation with the aflatoxigenic isolate increased corn grain aflatoxin levels by 188% compared to the non-inoculated control, while CT3 and K49 inoculation reduced aflatoxin levels in corn grain by 86 and 60%, respectively. In 2002, the non-toxigenic CT3 and K49 reduced aflatoxin levels by 61 and 76% compared to non-inoculated controls, respectively. In 2001, mixtures of aflatoxigenic and non-aflatoxigenic isolates had little effect on aflatoxin levels, but in 2002, inoculation with mixtures of K49 and CT3 reduced aflatoxin levels 68 and 37% compared to non-inoculated controls, respectively. In 2003 and 2004, a low level of natural aflatoxin contamination was observed (8 ng g−1). However, inoculation with mixtures of K49 + F3W4 and CT3 + F3W4, reduced levels of aflatoxin 65–94% compared to the aflatoxigenic strain alone. Compared to the non-sclerotia producing CT3, strain K49 produces large sclerotia, has more rapid in vitro radial growth, and a greater ability to colonize corn when artificially inoculated, perhaps indicating greater ecological competence. Results indicate that non-aflatoxigenic, indigenous A. flavus isolates, such as strain K49, have potential use for biocontrol of aflatoxin contamination in southern US corn.

Ecology of Aspergillus flavus, regulation of aflatoxin production, and management strategies to reduce aflatoxin contamination of corn

The contamination of corn (maize) by fungi and the accumulation of mycotoxins are a serious agricultural problem for human and animal health. One particular devastating group of mycotoxins, called aflatoxins, has been intensely studied since the 1960s. Studies of Aspergillus flavus, the agriculturally relevant producer of aflatoxins, have led to a well-characterized biosynthetic pathway for aflatoxin production, as well as a basic understanding of the organism’s life cycle. Unfortunately, these efforts have not resulted in corn production practices that substantially reduce aflatoxin contamination. Similarly, the use of agrochemicals (e.g., fungicides) results in very limited reduction of the fungus or the toxin. Thus, cultural management (fertility and irrigation) coupled with aggressive insect management is current recommendation for integrated aflatoxin management. The development of resistant hybrids appears to be a very promising technology, but commercial hybrids are still not available. Thus, biocontrol appears to be the most promising available avenue of reducing aflatoxin accumulation. Biocontrol utilizes nontoxigenic strains of Aspergillus to reduce the incidence of toxin-producing isolates through competitive displacement. To maximize the effectiveness of biocontrol, a thorough knowledge of the environmental factors influencing colonization and growth of Aspergillus is needed. A. flavus not only colonizes living plant tissue, but it also grows saprophytically on plant tissue in the soil. These residues serve as a reservoir for the fungus, allowing it to overwinter, and under favorable conditions it will resume growth and release new conidia. The conidia can be transmitted by air or insects to serve as new inoculum on host plants or debris in the field. This complex ecology of Aspergilli has been studied, but our understanding lags behind what is known about biosynthesis of the toxin itself. Our limited understanding of Aspergilli soil ecology is in part due to limitations in evaluating Aspergilli, aflatoxin, and the biosynthetic genes in the varying aspects of the environment. Current methods for assessing Aspergillus and aflatoxin accumulation rely heavily on cultural and analytical methods that are low throughput and technically challenging. Thus to understand Aspergillus ecology and environmental effects in contamination to maximize biocontrol efforts, it is necessary to understand current treatment effects and to develop methodologies capable of assessing the fungal populations present. In this manuscript we discuss the current knowledge of A. flavus ecology, the application of selected molecular techniques to field assessments, and crop practices used to reduce aflatoxin contamination, focusing on chemical treatments (fungicides and herbicides), insect management, and crop management.

Structure-dependent phytotoxicity of fumonisins and related compounds in a duckweed bioassay

Abstract Fumonisins A1, A2, B2, B2, and B3 are a series of secondary metabolite analogues produced by Fusarium moniliforme. The A series fumonisins differ from B series by possession of a N terminal acetyl group. Hydrolytic removal of the propanetricarboxylic acid moieties from fumonisins B1, and B2 yields the aminoalcohols HB1, and HB2, respectively. AAL-toxin is a chemically related phytotoxin (only one propanetricarboxylic acid) produced by Alternaria alternata. In a duckweed (Lemna pausicostata) bioassay, AAL-toxin and the B series fumonisins at 1,μM caused pronounced cellular leakage of electrolytes and photodegradation of chlorophylls. These four compounds also caused the most marked reductions in duckweed growth. HB1, at 1 μM was a moderate growth inhibitor and caused a low level of cellular leakage. The other compounds were inactive at this concentration. Significant effects on cellular leakage were measured at 0.04 μM for both AAL-toxin and fumonisin B1, the two most active analogues. The propanetricarboxylic acid groups of fumonisins and AAL-toxin are necessary for significant herbicidal activity of this series of compounds, whereas acetylation of the terminal amine group greatly reduces their activity. The structurally related sphingolipids, phytosphingosine and sphingosine, were about two orders of magnitude less phytotoxic than fumonisin B1; however, the phytotoxicity symptoms were similar.

FvVE1 regulates biosynthesis of the mycotoxins fumonisins and fusarins in Fusarium verticillioides.

The veA gene positively regulates sterigmatocystin production in Aspergillus nidulans and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus . Whether veA homologues have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homologue, FvVE1, on the production of two mycotoxin families, fumonisins and fusarins, in the important corn pathogen Fusarium verticillioides . We found that FvVE1 deletion completely suppressed fumonisin production on two natural substrates, corn and rice. Furthermore, our results revealed that FvVE1 is necessary for the expression of the pathway-specific regulatory gene FUM21 and structural genes in the fumonisin biosynthetic gene (FUM) cluster. FvVE1 deletion also blocked production of fusarins. The effects of FvVE1 deletion on the production of these toxins were found to be the same in two separate mating types. Our results strongly suggest that FvVE1 plays an important role in regulating mycotoxin production in F. verticillioides .

Natural Occurrence of Fumonisins in Rice with Fusarium Sheath Rot Disease

Twenty samples of rough rice (Oryza sativa) (unpolished kernels) collected during the 1995 harvest season from Arkansas (seven samples) and Texas (13 samples) were obtained from rice fields known to include plants with symptoms of Fusarium sheath rot putatively caused by Fusarium proliferatum. Samples were analyzed for fumonisin B1 (FB1) at three laboratories using three different extracting solvents by high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA) methods. Forty percent of the samples were positive for FB1 at levels ≤4.3 μg/g by HPLC. The same samples contained FB1 at ≤3.6 μg/g when measured by an ELISA method. Most samples that were positive for FB1 were positive for fumonisin B2 (FB2) and fumonisin B3 (FB3) by HPLC at levels ≤1.2 μg/g. Very good agreement was obtained among the two laboratories using HPLC methods and the third using ELISA. Shelling of the unpolished rice results in hull and brown rice fractions. In a sample that contained 4.3 μg/g in whole kernels, the fumonisin level was very high in hulls (≤16.8 μg/g) and low in brown rice (≤0.9 μg/g). Milling of brown rice results in bran and white rice fractions. Fumonisins were found in bran at a level of ≤3.7 μg/g but were below the level of detection by HPLC in white rice. The presence of fumonisins (FB1, FB2, and FB3) was confirmed by fast atom bombardment/mass spectrometry. This is the first report of fumonisins in naturally contaminated rice in the United States.

Relationships between aflatoxin production and sclerotia formation among isolates of Aspergillus section Flavi from the Mississippi Delta

Aspergillus section Flavi isolates, predominately A. flavus, from different crops and soils differed significantly in production of aflatoxin and sclerotia. About 50% of the isolates from corn, soil and peanut produced large sclerotia, while only 20% of the rice isolates produced large sclerotia. There was a higher frequency of small sclerotia-producing isolates from rice compared to the other sources and isolates that did not produce sclerotia were significantly less likely to be toxigenic than strains that produced large sclerotia.

Phytotoxicity and mammalian cytotoxicity of macrocyclic trichothecene mycotoxins from Myrothecium verrucaria

Macrocyclic trichothecene toxins produced by Myrothecium verrucaria (a phytopathogen of interest in biological weed control) and the non-trichothecene toxin atranone B from Stachybotiys atra were tested for phytotoxicity in duckweed (Lemna pausicostata L.) plantlet cultures and kudzu (Pueraria lobata L.) leaf disc assays, and for mammalian cytotoxicity in four cultured cell lines. Roridin E and H, epi-isororidin E, and verrucarin A and J were phytotoxic (half-maximal effect in the concentration range 0.1-9.7 microM on duckweed and 1.5->80 microM on kudzu) and cytotoxic to mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 1-35 nM). Trichoverrins A and B and atranone B were moderately phytotoxic (half-maximal effect in the concentration range 1 9-69 microM on duckweed and 13->80 microM on kudzu) and weakly cytotoxic with mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 0.3->2 microM).

Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize

Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 and 2009 to assess the competitiveness of non-aflatoxigenic strains when challenged against toxigenic strains using a pin-bar inoculation technique. In three sets of experiments the non-aflatoxigenic strain K49 effectively displaced toxigenic strains at various concentrations or combinations. The fourth study compared the relative competitiveness of three non-aflatoxigenic strains (K49, NRRL 21882 from Afla-Guard®, and AF36) when challenged on maize against two aflatoxin- and cyclopiazonic acid (CPA)-producing strains (K54 and F3W4). These studies indicate that K49 and NRRL 21882 are superior to AF36 in reducing total aflatoxin contamination. Neither K49 nor NRRL 21882 produce CPA and when challenged with K54 and F3W4, CPA and aflatoxins were reduced by 84–97% and 83–98%, respectively. In contrast, AF36 reduced aflatoxins by 20% with F3W4 and 93% with K54 and showed no reduction in CPA with F3W4 and only a 62% reduction in CPA with K54. Because AF36 produces CPA, high levels of CPA accumulate when maize is inoculated with AF36 alone or in combination with F3W4 or K54. These results indicate that K49 may be equally effective as NRRL 21882 in reducing both aflatoxins and CPA in maize.