Aflatoxins are a secondary metabolite produced by the fungus Aspergillus flavus. A. flavus has been known to infect several crops including tree nuts, peanuts, rice, cotton and maize. Aflatoxins have been found to cause tumors with aflatoxin B1 being the most carcinogenic biologically produced substance known to man. Therefore, the FDA has restricted the amount of aflatoxin in maize for human consumption to 20 ppb (ng/g). An estimated $225 million are lost each year in the United States due to aflatoxin contamination in maize crops alone. Agriculture is a vital part of Mississippi’s economy, and maize is one of its largest crops. The purpose of this research is to track the correlations between aflatoxin accumulation and Aspergillus flavus fungal biomass for the first several weeks after inoculation, as well as the spreading of the fungus and the aflatoxin throughout the inoculated ear of maize. This will allow for better understanding of the pathogen-host interactions and how the fungus progresses over time. GA209 x T173 is the aflatoxin accumulation susceptible maize hybrid, GA209 x Mp313E is the susceptible and resistant hybrid, and Mp717 x Mp313E is the resistant maize hybrid to aflatoxin accumulation. These maize hybrids were each inoculated with toxin producing Aspergillus flavus NRRL 3357 and water as a control 21 days after silk maturation. Collections of the inoculated maize cobs were made 3, 7, 14, 21, 28, 35, and 60 days after inoculation. Maize samples were collected and analyzed for aflatoxin and DNA concentration. The extracted aflatoxin was analyzed using an LC/MS. The fungal biomass was determined by performing quantitative real time polymerase chain reaction (PCR). GA209xT173 and Mp717xMp313E showed no aflatoxin production two days after inoculation. The resistant maize hybrid lead in aflatoxin accumulation the last two years but had the least amount of fungal biomass for second and third years of the experiment The production of aflatoxin seems to begin decelerating after 21 days after inoculation. Resistance characteristics are more to prevent fungal infection. Fungal biomass was significantly higher in the susceptible hybrid GA209xT173 compared to the other hybrids. However, fungal spread was significantly higher in Mp313ExT173 and Mp717xMp313E.

by Chan Hoi Tak. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 129-137). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Contents --- p.viii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Aflatoxins --- p.1 / Chapter 1.1.1 --- AFB1 Metabolism --- p.5 / Chapter 1.1.2 --- Bioactivation --- p.5 / Chapter 1.1.3 --- Detoxification --- p.8 / Chapter 1.1.4 --- Toxicity of AFB1 --- p.9 / Chapter 1.2 --- Licorice Plants --- p.11 / Chapter 1.2.1 --- Glycyrrhizic Acid (GA) --- p.13 / Chapter 1.2.2 --- Metabolism of GA --- p.16 / Chapter 1.2.3 --- Adverse Effects of GA --- p.19 / Chapter 1.3 --- Aim of Research --- p.19 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.21 / Chapter 2.1 --- Cell cultures --- p.21 / Chapter 2.1.1 --- Isolation of splenocytes --- p.21 / Chapter 2.1.2 --- Culture of cell lines --- p.22 / Chapter 2.1.3 --- Trypsinziation of cells --- p.22 / Chapter 2.2 --- Preparation of drugs --- p.23 / Chapter 2.2.1 --- Preparation of commercially available chemicals for cell culture --- p.23 / Chapter 2.2.2 --- Preparation of Licorice extracts for cell culture --- p.23 / Chapter 2.2.3 --- Preparation of chemicals for enzymatic reaction --- p.24 / Chapter 2.3 --- Cytotoxicity assay Methods --- p.24 / Chapter 2.3.1 --- MTT assay --- p.25 / Chapter 2.3.2 --- Neutral red assay --- p.25 / Chapter 2.4 --- Cytotoxicity Assay --- p.32 / Chapter 2.5 --- Chemoprotection assay --- p.32 / Chapter 2.5.1 --- Direct addition of drugs --- p.33 / Chapter 2.5.2 --- Pretreatment of cells --- p.33 / Chapter 2.5.3 --- Culture of cells with drugs --- p.34 / Chapter 2.6 --- Preparation of enzymes --- p.36 / Chapter 2.6.1 --- Preparation of cells --- p.36 / Chapter 2.6.2 --- Pretreatment of rat microsomes --- p.36 / Chapter 2.7 --- Bradford Assay --- p.37 / Chapter 2.8 --- Alkoxyresorufin O-Dealkylase assay --- p.39 / Chapter 2.8.1 --- Effects of AFB1 on P450 --- p.44 / Chapter 2.8.2 --- Effects of drugs on P450 --- p.44 / Chapter 2.9 --- Glutathione-S-transferase assay --- p.44 / Chapter 2.9.1 --- GST assay in rat microsomal fractions --- p.48 / Chapter 2.9.1.1 --- Effects of AFB1 on GST --- p.48 / Chapter 2.9.1.2 --- Effects of drugs on GST --- p.48 / Chapter 2.9.2 --- GST assay in crude fractions from cells --- p.48 / Chapter 2.9.2.1 --- Direct addition effects of drugs on GST --- p.48 / Chapter 2.9.2.2 --- Effects of drug-containing medium on GST --- p.49 / RESULTS / Chapter CHAPTER THREE --- CELL CULTURES --- p.50 / Chapter 3.1 --- Cytotoxicity assay --- p.51 / Chapter 3.1.1 --- Cytotoxicity of AFB1 --- p.51 / Chapter 3.1.1.1 --- Cytotoxicity of AFB1 to splenocytes --- p.51 / Chapter 3.1.1.2 --- Cytotoxicity of AFB1 to cell lines --- p.57 / Chapter 3.1.2 --- Cytotoxicity of drugs to cell lines --- p.62 / Chapter 3.2 --- Chemoprotection assay --- p.73 / Chapter 3.2.1 --- Direct addition of drugs --- p.73 / Chapter 3.2.2 --- Pretreatment of cells with drugs --- p.78 / Chapter 3.2.3 --- Culture of cells with drugs --- p.83 / Chapter CHAPTER FOUR --- ENZYMATIC ASSAYS --- p.87 / Chapter 4.1 --- Alkoxyresorufin O-dealkylase assay --- p.88 / Chapter 4.1.1 --- Effects of AFB1 on P450s --- p.88 / Chapter 4.1.2 --- Effects of GA on P450s --- p.92 / Chapter 4.1.3 --- Effects of EX on P450s --- p.95 / Chapter 4.2 --- Glutathione -S- transferase assay --- p.98 / Chapter 4.2.1 --- Effects of drugs on GST in cells --- p.100 / Chapter 4.2.2 --- Effects of AFB1 and drugs on GST from rats --- p.108 / Chapter 4.2.2.1 --- Effects of AFB1 on GST from rats --- p.108 / Chapter 4.2.2.2 --- Effects of drugs on GST from rats --- p.112 / Chapter CHAPTER FIVE --- DISCUSSION --- p.115 / Chapter 5.1 --- Cytotoxicity of AFB1 --- p.118 / Chapter 5.2 --- "Cytotoxicity of EX, GA and AA" --- p.119 / Chapter 5.3 --- Chemoprotection of GA and EX on AFB1 cytotoxicity --- p.121 / Chapter 5.4 --- Effects of GA and EX on enzymes involved in AFB1 metabolism --- p.123 / REFERENCES --- p.129

Aflatoxins are potent carcinogens produced by the fungus Aspergillus flavus Link:Fr and are a significant preharvest problem in maize production in Texas, the southern US, and subtropical climates. Several sources of maize germplasm are available which reduce preharvest aflatoxin accumulation, but many of these sources lack agronomic performance for direct use as a parent in commercial hybrids. Tropical germplasm is a source of both resistance to aflatoxin accumulation resistance and agronomic performance traits. The goal of this study was to investigate germplasm for traits to reduce preharvest aflatoxin accumulation. The specific objectives of this research were: 1) to validate QTL estimates previously identified in lines per se and estimate new QTL associated with reduced aflatoxin accumulations and agronomic traits; 2) to evaluate agronomic characteristics of selections from a RIL population in testcrosses at multiple locations across Texas; and (3) to release agronomically desirable germplasm sources with reduced risk to preharvest aflatoxin accumulation. A total of 96 QTLs were detected across fourteen measured traits using an RIL population of 130 individuals in testcross hybrids evaluated in five environments. Three QTL detected in per se analyses were also detected in hybrid testcrosses. Previously unreported QTL were detected on chromosomes 3, 4, 8 and 9. Within each of the two years, neither subset of the RIL testcross hybrids produced grain yields equal to commercial hybrid checks in these trials, but one testcross in 2008 produced grain yield within 10 percent of commercial check hybrids and in 2009, five RIL testcrosses produced grain yield within 17 percent of the commercial check hybrids. Although RIL testcrosses did not yield more than the commercial checks, they will be a source of germplasm for reduced aflatoxin. Improved sources of maize germplasm lines Tx736, Tx739, and Tx740 have been selected for adaptation to southern US and Texas growing environments with traits that reduce aflatoxin accumulation. Each of the lines in testcross accumulated significantly fewer aflatoxins than commercial hybrids in the trial.

The diploma thesis "Determination of aflatoxin in selected products" deals mainly with aflatoxin, its occurrence in raw materials of plant origin and methods of decontamination. The studied toxin is produced by fibrous micromycetes primarily from genus Aspergillus is their secondary metabolites. It is very widespread and is a frequent cause of retention of raw materials at the border. Factors that affect production of aflatoxin are, among other things, water activity and pH and are also determined in samples and mentioned in theoretical part. A total of 30 samples were tested for aflatoxin B1, including batch of apricot jam, two batches of Svatojánské ořechy and two batches of Jarní hustopečské mandle. A heterogeneous competitive enzyme immunoassay (ELISA) was used for the analysis. Detectable amounts of AFB1 in the range of 1,26 - 1,66 μg∙kg-1 showed 26,67 % of samples. These values were determined in samples of Svatojánské ořechy and Jarní hustopečské mandle, the apricot jam did not contain measurable amount of AFB1.

Aflatoxins are naturally occurring carcinogens found on grains, particularly in warmer climates. Because of their carcinogenic properties, they are strictly regulated and are only allowed in minimal amounts. Aflatoxin B1, the most potent naturally occurring carcinogen known, is metabolized in the liver to form aflatoxin M1, which is present in the milk of lactating animals. If aflatoxin concentrations are elevated above legal limits, the milk cannot be used for human consumption. Because of this, research has been conducted to evaluate ways to mitigate its absorption in the animal and prevent transfer to the milk. One such way is through the use of clay adsorbents. The current studies aimed to evaluate the efficacy of two different clay adsorbents at preventing aflatoxin transfer to the milk of Holsteins fed a known concentration of aflatoxin.

Ethanol fermentation of highly concentrated aflatoxin-contaminated corn (Zea mays) was conducted on a lab scale to determine if aflatoxin concentrated in the distilled ethanol and/or dry distillers grain end-products. Alliquots of fermented mash, distilled ethanol, stillage, and dry distillers grain (DDG) were analyzed via LC-MS/MS and immunoassay detection methods for aflatoxin. Results indicate that aflatoxin does not greatly concentrate during fermentation in the DDGs and is undetectable in distilled ethanol. Addition of binders, MTB-100®, to aflatoxin-contaminated DDGs showed great reduction in aflatoxin concentrations when analyzed via LC-MS/MS. Also, an experiment investigating detoxification of aflatoxin using Clorox® was conducted. Results obtained from LC-MS/MS showed a positive correlation of decreased aflatoxin levels with increasing Clorox® levels following a logarithmic trend.

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DESIGN, SYNTHESIS, AND EVALUATION OF DITERPENONES AS POTENT CHEMOPREVENTIVE AGENTS FOR AFB1 INDUCED CARCINOGENISIS IN HUMAN LIVER CELLS By Miguel A. Zuniga, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Major Director: Qibing Zhou, Ph.D Assistant Professor Department of Chemistry Terpene quinone methides (TPQMs) have been isolated from a variety of plants and show broad activities against bacteria, fungi, and cancerous cell lines. The biological activity has been attributed to the reactive electrophilic QM moiety, this structural feature has long been recognized as an intermediate in organic synthesis and in certain biosynthetic processes. It has been shown that quinone methide structures play a key role in the chemistry of several classes of antibiotic drugs and antitumor compounds such as mitomycin C and anthracyclines. The goal of this study was to understand the basis of QM bioactivity so that terpene catechols as analogs of natural TPQMs precursors can be designed as effective chemopreventive agents.In order to investigate the oxidation pathway of terpene QM precursors, a homoconjugated diterpene catechol was synthesized. A review of the literature revealed that Cu2+ -induced oxidation of simple catechols proceeds through a two-step one electron transfer process, and o-quinone is the sole oxidation product. In contrast, our studies showed direct p-QM formation from a diterpene catechol and no o-quinone oxidation products were observed. Furthermore, the Cu2+-induced oxidation pathway of our homoconjugated diterpene catechol revealed multiple p-QM formations under aqueous conditions. The implications of these findings suggest that terpene QM precursors can cause extensive DNA damage through in situ generated hydroxyl radicals or by DNA alkylations with p-QMs. To elucidate the Cu2+-induced DNA damage mechanism, a series of catechol analogues of natural terpene QM precursors were designed to investigate potential effects of stereochemistry, substitutiional, and functional groups on nucleobase alkylation and production of reactive oxygen species. The results of these tests suggested that production of ROS was the dominant mechanism for the observed DNA damage in the Cu2+-induced oxidation regardless of stereo and structural differences of catechols or subsequent oxidation products as QM or quinone. From the DNA damage study we found that the presence of NADH significantly enhanced the extent of DNA damage by oxidation of these catechols. More specifically, in the case of alkene catechols, our results showed that DNA damage was independent of the concentration of catechols, thus providing ample evidence for production of ROS through the redox cycle of catechols/quinones. Additional support for the formation of hydroxyl radical and futile redox cycling was clearly demonstrated by comparison of the fragmentation pattern with that of a Fenton reaction. The identify of the ROS was also shown to be in the form of a Cu(I)OOH complex by radical scavenging and metal chelation experiments. Cis-terpenones were first shown to have chemoprotective activity by Dr. Zhou and colleagues. In collaboration with their efforts to identify the mode of action of cis-terpenones, another project to achieve an isotope labeled cis-terpenone was undertaken. The isotope study was employed to obtain and experimentally demonstrate the feasibility of incorporating a radioactive label in cis-terpenone for the future studies of cis-terpenone metabolism. An analysis of the deuterium labeled cis-terpenone from the isotope exchange reaction showed that the isotope was being incorporated into multiple positions through scattering processes. This non-radioactive isotope study made it possible to optimize the conditions prior to using a radioactive tritium label, which will be a requisite for future metabolic studies.As an extension of this work, a structure activity relationship (SARs) study was undertaken with a focus on improving the physiochemical properties and chemical stability of cis-terpenones. The primary purpose of this study was to attempt to explain the reason for the observed protective effects of cis-terpenones against AFB1. Considerable efforts were made to introduce an unsaturated double bond in the structure of cis-terpenone by an intramolecular Pd (II) catalyzed Heck reaction. Unfortunately, this method was unsuccessful and which was attributed to the disconnection of our starting material during the formation of an enolate intermediate. A second model study to generate desired coumarin and ditepene related structures was investigated with a Diels Alder [4+2] cycloaddition reaction. After numerous attempts, we found that the successfull synthesis of these compounds was highly dependent on the temperature, solvent, and the use of stabilizers in the reaction. Finally, the targeted diterpene analogs were screened for protective effects against AFB1 by the MTT cell viability assay. However, these preliminary data showed that additional structural features and key modifications are still required to better correlate the structure with the mechanism of chemoprotection.