A Public Platform for the Verification of the Phenotypic Effect of Candidate Genes for Resistance to Aflatoxin Accumulation and Aspergillus flavus Infection in Maize

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline.     

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard^® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.     

Loss of msnA, a Putative Stress Regulatory Gene, in Aspergillus parasiticus and Aspergillus flavus Increased Production of Conidia, Aflatoxins and Kojic Acid

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.     

Screening a Strain of Aspergillus niger and Optimization of Fermentation Conditions for Degradation of Aflatoxin B1

Aflatoxin B₁, a type of highly toxic mycotoxin produced by some species belonging to the Aspergillus genus, such as Aspergillus flavus and Aspergillus parasiticus, is widely distributed in feed matrices. Here, coumarin was used as the sole carbon source to screen microorganism strains that were isolated from types of feed ingredients. Only one isolate (ND-1) was able to degrade aflatoxin B₁ after screening. ND-1 isolate, identified as a strain of Aspergillus niger using phylogenetic analysis on the basis of 18S rDNA, could remove 26.3% of aflatoxin B₁ after 48 h of fermentation in nutrient broth (NB). Optimization of fermentation conditions for aflatoxin B₁ degradation by selected Aspergillus niger was also performed. These results showed that 58.2% of aflatoxin B₁ was degraded after 24 h of culture under the optimal fermentation conditions. The aflatoxin B₁ degradation activity of Aspergillus niger supernatant was significantly stronger than cells and cell extracts. Furthermore, effects of temperature, heat treatment, pH, and metal ions on aflatoxin B₁ degradation by the supernatant were examined. Results indicated that aflatoxin B₁ degradation of Aspergillus niger is enzymatic and this process occurs in the extracellular environment.     

Molecular Mechanisms of Lipoic Acid Protection against Aflatoxin B1-Induced Liver Oxidative Damage and Inflammatory Responses in Broilers

Alpha-lipoic acid (α-LA) was evaluated in this study for its molecular mechanisms against liver oxidative damage and inflammatory responses induced by aflatoxin B₁ (AFB₁). Birds were randomly allocated into four groups with different diets for three weeks: a basal diet, a 300 mg/kg α-LA supplementation in a basal diet, a diet containing 74 μg/kg AFB₁, and 300 mg/kg α-LA supplementation in a diet containing 74 μg/kg AFB₁. In the AFB₁ group, the expression of GSH-P_X mRNA was down-regulated (p < 0.05), and the levels of lipid peroxide and nitric oxide were increased (p < 0.05) in the chicken livers compared to those of the control group. Additionally, the mRNA level of the pro-inflammatory factor interleukin-6 was up-regulated significantly (p < 0.05), the protein expressions of both the nuclear factor kappa B (NF-κB) p65 and the inducible nitric oxide synthase were enhanced significantly (p < 0.05) in the AFB₁ group. All of these negative effects were inhibited by α-LA. These results indicate that α-LA may be effective in preventing hepatic oxidative stress, down-regulating the expression of hepatic pro-inflammatory cytokines, as well as inhibiting NF-κB expression.     

Antimelanoma Potential of Eruca sativa Seed Oil and its Bioactive Principles

The present communication reports the comparison of in vivo antioxidant, antimelanoma and antimutagenic activities of Eruca sativa seed oil and its bio principles (allyl isothiocyanate, phenylethyl isothiocyanate and sulphoraphane) against B16F10 melanoma cells induced in C57BL/6 mice model. Among the various treatments considered for the study, isothiocyanates combination (allyl isothiocyanate, phenylethyl isothiocyanate and sulphoraphane; 1:1:1; 10 µM) exhibited optimum antioxidant activity, 51.95±1.14 µM glutathione per mg protein compared to seed oil 25.91±1.26 µM. Lipid peroxidation value was 9.97±1.72 µM malondialdehyde per mg wet weight for isothiocyanates combination against seed oil, 28.45±1.87 µM and rendered significant protection against oxidative stress induced by melanoma in liver tissue. Isothiocyanates combination significantly suppressed various parameters, such as tumor growth, isothiocyanates combination by 36.36% while the seed oil by 15.23%; tumor weight, isothiocyanates combination by 45.9% and seed oil by 19.6%; tumor volume, isothiocyanates combination by 41.7% while the seed oil by 32.3%, measured for antimelanoma activity at a concentration of 10 µM. Isothiocyanates combination has been found to be more cytotoxic bioagent against B16F10 melanoma cells induced in C57BL/6 mice compared to naturally occurring Eruca sativa seed oil.     

Oxathiolene oxides: a novel family of compounds that induce ferritin,glutathione S-transferase,and other proteins of the phase II response

Compounds that induce the synthesis of cytoprotective phase II enzymes have shown promise as cancer chemopreventive agents. Although chemically diverse, phase II enzyme inducers are capable of participating in Michael reaction chemistry. We have synthesized a novel class of organosulfur compounds, termed oxathiolene oxides (OTEOs). Based on their chemical properties, we hypothesized that these compounds could function as phase II enzyme inducers. Northern blot analysis showed that oxathiolene oxides induce the phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and ferritin H and L mRNA in a concentration-dependent fashion in a normal embryonic mouse liver cell line, BNLCL.2. OTEO-562 (3-cyclohexenyl-4-methyl-1,2-oxathiol-3-ene-2-oxide) was the strongest inducer. Western blot analysis demonstrated that GST-alpha and ferritin H protein levels were also induced in cells treated with OTEO-562, as was total GST and NQO1 enzyme activity. Further, induction of NQO1 activity by OTEO-562 was equivalent in aromatic hydrocarbon (Ah) receptor wild-type and Ah receptor mutant cell lines, suggesting that oxathiolene oxides activate phase II enzymes by an Ah receptor-independent mechanism. Consistent with this observation, OTEO-562 failed to induce cytochrome P450 1A1 mRNA. These results suggest that oxathiolene oxides may merit further investigation as candidate chemopreventive agents.     

CDA-II,a urinary preparation,induces growth arrest and apoptosis of human leukemia cells through inactivation of nuclear factor-kappaB in a caspase-dependent manner

CDA-II (cell differentiation agent II) was a urinary preparation, isolated from healthy human urine. We determined the anticancer activity of CDA-II using human acute myeloid leukemia (AML) cell lines, K562, Kasumi-1 and KG-1. An in vitro cytotoxicity assay showed that CDA-II exhibited growth arrest in leukemic cells, while it did not induce cytotoxicity in normal peripheral blood monouclear cells (PBMCs). In vivo studies using the Kasumi-1 xenografted SCID mouse model showed tumor inhibition rate were increased and the survival time were prolonged in a dose-dependent manner, without any significant toxicity on mice body. Depolarized mitochondrial membranes and the activation of caspase-3, 9 as well as PARP were found in leukemic cells treated with CDA-II for 6–24 h. We further found NF-κB nuclear translocation were prevented by CDA-II treatment, which therefore inactivated NF-κB and down-regulated its target genes expression, including Bcl-2/Bax ratio, Mcl-1 and XIAP. The caspase-3 inhibitor Z-DEVD-FMK inhibited CDA-II-induced apoptosis and CDA-II combined with NF-κB inhibitor PDTC significantly increased the apoptotic rate of leukemic cells. We concluded that CDA-II potently induced caspase-dependent leukemia-specific apoptosis in leukemic cells mediated through inactivation of NF-κB, involving in Bcl-2 family and XIAP, which has no cytotoxicity on normal cells.     

Opportunities in discovery and delivery of anticancer drugs targeting mitochondria and cancer cell metabolism

Cancer cells are characterized by self-sufficiency in the absence of growth signals, their ability to evade apoptosis, resistance to anti-growth signals, sustained angiogenesis, uncontrolled proliferation, and invasion and metastasis. Alterations in cellular bioenergetics are an emerging hallmark of cancer. The mitochondrion is the major organelle implicated in the cellular bioenergetic and biosynthetic changes accompanying cancer. These bioenergetic modifications contribute to the invasive, metastatic and adaptive properties typical in most tumors. Moreover, mitochondrial DNA mutations complement the bioenergetic changes in cancer. Several cancer management therapies have been proposed that target tumor cell metabolism and mitochondria. Glycolytic inhibitors serve as a classical example of cancer metabolism targeting agents. Several TCA cycle and OXPHOS inhibitors are being tested for their anticancer potential. Moreover, agents targeting the PDC/PDK (pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase) interaction are being studied for reversal of Warburg effect. Targeting of the apoptotic regulatory machinery of mitochondria is another potential anticancer field in need of exploration. Additionally, oxidative phosphorylation uncouplers, potassium channel modulators, and mitochondrial redox are under investigation for their anticancer potential. To this end there is an increased demand for agents that specifically hit their target. Delocalized lipophilic cations have shown tremendous potential in delivering anticancer agents selectively to tumor cells. This review provides an overview of the potential anticancer agents that act by targeting cancer cell metabolism and mitochondria, and also brings us face to face with the emerging opportunities in cancer therapy.