Carol H. Carter-Wientjes

Antiestrogenic glyceollins suppress human breast and ovarian carcinoma tumorigenesis

Purpose: We have identified the phytoalexin compounds glyceollins I, II, and III, which exhibit marked antiestrogenic effects on estrogen receptor function and estrogen-dependent tumor growth in vivo. The purpose of this study was to investigate the interactions among the induced soy phytoalexins glyceollins I, II, and III on the growth of estrogen-dependent MCF-7 breast cancer and BG-1 ovarian cancer cells implanted in ovariectomized athymic mice. Experimental Design: Four treatment groups for each cell line were used: vehicle control, 20 mg/kg/mouse/d glyceollin mixture injection, 0.72 mg estradiol (E2) implant, and E2 implant + 20 mg/kg/mouse/d glyceollin injection. Results: Treatment with glyceollin suppressed E2-stimulated tumor growth of MCF-7 cells (−53.4%) and BG-1 cells (−73.1%) in ovariectomized athymic mice. These tumor-inhibiting effects corresponded with significantly lower E2-induced progesterone receptor expression in the tumors. In contrast to tamoxifen, the glyceollins had no estrogen-agonist effects on uterine morphology and partially antagonized the uterotropic effects of estrogen. Conclusions: These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma.

Effect of soybean volatile compounds on Aspergillus flavus growth and aflatoxin production.

Soybean homogenates produced volatile compounds upon exposure to lipase. These induced volatiles were identified by SPME. Seventeen volatile compounds identified by SPME were chosen for determination of their ability to inhibit Aspergillus flavus growth and aflatoxin B(1) (AFB1) production in a solid media assay. These volatiles included aldehydes, alcohols, ketones, and furans. Of the tested compounds, the aldehydes showed the greatest inhibition of fungal growth and AFB1 production. These compounds inhibited up to 100% of the observed growth and AFB1 production as compared to the controls. The greatest activity by the aldehydes to disrupt growth was ranked as follows: 2,4 hexadienal > benzaldehyde > 2-octenal > (E)-2-heptenal > octanal > (E)-2-hexenal > nonanal > hexanal. The greatest activity by the aldehydes to reduce AFB1 was ranked as follows: (E)-2-hexenal > 2,4 hexadienal > (E)-2-heptenal > hexanal > nonanal. (E)-2-hexenal and (E)-2-heptenal were tested further in an A. flavus-inoculated corn kernel assay. Both compounds prevented colonization by A. flavus and eliminated AFB1 production when exposed to compound volumes < 10 muL as also shown in the solid media assay. The results suggest that soybeans react to lipase by production of potent antifungal volatiles.

Estrogenic and Antiestrogenic Activities of Phytoalexins from Red Kidney Bean (Phaseolus vulgaris L.)

Legumes are the predominant source of isoflavones considered to be phytoestrogens that mimic the hormone 17β-estradiol (E2). Due to the risks associated with hormone replacement therapy, there is a growing need for alternative sources of estrogenic formulations for the treatment of menopausal symptoms. Legume phytoalexins (induced isoflavones) are produced under conditions of stress that include insect damage, wounding, or application of elicitors. The estrogenic and antiestrogenic activities of methanolic extracts obtained from red kidney bean treated with the fungus Aspergillus sojae were compared with those of untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay. A. sojae-treated red kidney bean extracts displayed both estrogenic and antiestrogenic activities. Analysis of elicitor-treated red kidney bean extracts showed that A. sojae treatments achieved maximal levels of kievitone at 1199 ± 101 μg/g and phaseollin at 227.8 ± 44 μg/g. The phytoalexins kievitone and phaseollin were isolated from A. sojae-treated red kidney bean extracts and analyzed for estrogenic activity using ERα and ERβ binding, ERE luciferase assays in MCF-7 and HEK 293 cells, and MCF-7 cell proliferation. Kievitone showed the highest relative binding affinity to ERα with kievitone (0.48%) > phaseollin (0.21%), and phaseollin showed the highest relative binding affinity to ERβ with phaseollin (0.53%) > kievitone (0.42%). In an ERE luciferase assay in MCF-7 cells, kievitone displayed high ER transactivation at 10 μM; phaseollin displayed low ER transactivation. Both kievitone and phaseollin stimulated MCF-7 cell proliferation, with kievitone displaying agonist activity between 0.1 and 10 μM. Cotransfection reporter assays performed in HEK 293 demonstrated that phaseollin selectively increased ERE transcriptional activity of ERβ and kievitone selectively increased ERE transcriptional activity of ERα. Although phaseollin displayed attenuation of ER transactivation in the ERE luciferase assay in MCF-7 cells, both phytoalexins attenuated the effects of E2 in an MCF-7 cell colonial survival assay. This work provides evidence that the red kidney bean phytoalexins kievitone and phaseollin possess both estrogenic and antiestrogenic activities.

Volatile Trans-2-Hexenal, a Soybean Aldehyde, Inhibits Aspergillus flavus Growth and Aflatoxin Production in Corn

UNLABELLED Trans-2-hexenal, a volatile aldehyde, is produced by soybean (Glycine max [L.] Merr) and other plants via the lipoxygenase pathway. In vitro tests showed it significantly (P < 0.001) reduced Aspergillus flavus germinating conidial viability at 10 μM, with approximately 95% viability reduction observed at 20 μM. The viability of nongerminated conidia was not reduced. To test the effectiveness of this volatile to prevent fungal growth in stored corn, trans-2-hexenal was pumped intermittently into glass jars containing corn. Experiments were performed to determine the ability of 2 different pump cycle time-courses to prevent A. flavus growth on sterile corn (23% moisture). Intermittently (30-min pumping period) over 7 d, this volatile was pumped through 350 g of corn kernels inoculated with 1 mL of 3 × 10⁴ conidia of A. flavus. Controls consisted of (1) sterile corn, (2) corn inoculated with A. flavus with no pumped air, and (3) corn inoculated with A. flavus with intermittently pumped air. Aflatoxin B₁ (AFB₁), viability counts, and aldehyde concentration in the headspace were performed in each experiment. To determine whether an increased time period between volatile pumping would prevent A. flavus growth, a 2nd series of experiments were performed that were similar to the 1st series except that trans-2-hexenal (only) was pumped for a 30-min period every 12 h. Experiments were performed 3 times for each time course. Both experiments showed that intermittent pumping of volatile trans-2-hexenal significantly (P < 0.001) prevented A. flavus growth and aflatoxin B₁ production over a 7-d period. PRACTICAL APPLICATION Results from this study indicate that intermittent pumping of volatile trans-2-hexenal could be used to protect stored corn from A. flavus growth and aflatoxin contamination.

The Aspergillus flavus Homeobox Gene, hbx1, Is Required for Development and Aflatoxin Production

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx) genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

Inhibition of Bacterial and Filamentous Fungal Growth in High Moisture, Nonsterile Corn with Intermittent Pumping of Trans-2-Hexenal Vapor

Trans-2-hexenal (T2H), a plant-produced aldehyde, was intermittently pumped over a 7 d period into a small, bench top model of stored corn (nonsterile, moisture content about 23%). Naturally occurring bacteria and fungi, including added Aspergillus flavus, grew rapidly on corn not treated with T2H vapor. However, intermittently pumped T2H (30 min per 2 h or 30 min per 12 h) significantly reduced bacterial and fungal viable populations, with nearly 100% fungal viability loss observed after either (1) one day of pumping at the 30 min per 2 h rate or (2) pumping cycles of 30 min per 12 h period over the initial 48 to 72 h of incubation. Data suggest that short-term intermittent fumigation of stored corn with T2H could prevent growth of bacteria and mycotoxigenic fungi such as A. flavus.

The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination

Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related (PR) proteins serve as an important defense mechanism against invading pathogens by conferring systemic acquired resistance in plants. Among these, production of the PR maize seed protein, ZmPRms (AC205274.3_FG001), has been speculated to be involved in resistance to infection by A. flavus and other pathogens. To better understand the relative contribution of ZmPRms to A. flavus resistance and aflatoxin production, a seed-specific RNA interference (RNAi)-based gene silencing approach was used to develop transgenic maize lines expressing hairpin RNAs to target ZmPRms. Downregulation of ZmPRms in transgenic kernels resulted in a ∼250–350% increase in A. flavus infection accompanied by a ∼4.5–7.5-fold higher accumulation of aflatoxins than control plants. Gene co-expression network analysis of RNA-seq data during the A. flavus-maize interaction identified ZmPRms as a network hub possibly responsible for regulating several downstream candidate genes associated with disease resistance and other biochemical functions. Expression analysis of these candidate genes in the ZmPRms–RNAi lines demonstrated downregulation (vs. control) of a majority of these ZmPRms-regulated genes during A. flavus infection. These results are consistent with a key role of ZmPRms in resistance to A. flavus infection and aflatoxin accumulation in maize kernels.

The Aspergillus flavus Spermidine Synthase (spds) Gene, Is Required for Normal Development, Aflatoxin Production, and Pathogenesis During Infection of Maize Kernels

Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as aflatoxins. Polyamines (PAs) are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put) by the enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc) and S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and aflatoxin production during seed infection. The data presented here suggest that future studies targeting spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce aflatoxin contamination in maize and possibly in other susceptible crops.