Shawna L. Lemke

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.

Absorption and retinol equivalence of β-carotene in humans is influenced by dietary vitamin A intake

The effect of vitamin A supplements on metabolic behavior of an oral tracer dose of [14C]β-carotene was investigated in a longitudinal test-retest design in two adults. For the test, each subject ingested 1 nmol of [14C]β-carotene (100 nCi) in an emulsified olive oil-banana drink. Total urine and stool were collected for up to 30 days; concentration-time patterns of [14C]β-carotene, [14C]retinyl esters, and [14C]retinol were determined for 46 days. On Day 53, the subjects were placed on a daily vitamin A supplement (10,000 IU/day), and a second dose of [14C]β-carotene (retest) was given on Day 74. All 14C determinations were made using accelerator mass spectrometry. In both subjects, the vitamin A supplementation was associated with three main effects: 1) increased apparent absorption: test versus retest values rose from 57% to 74% (Subject 1) and from 52% to 75% (Subject 2); 2) an ∼10-fold reduction in urinary excretion; and 3) a lower ratio of labeled retinyl ester/β-carotene concentrations in the absorptive phase. The molar vitamin A value of the dose for the test was 0.62 mol (Subject 1) and 0.54 mol (Subject 2) vitamin A to 1 mol β-carotene. Respective values for the retest were 0.85 and 0.74. These results show that while less cleavage of β-carotene occurred due to vitamin A supplementation, higher absorption resulted in larger molar vitamin A values.

Development and Characterization of a Carbon-Based Composite Material for Reducing Patulin Levels in Apple Juice

Patulin, a heterocyclic lactone produced by various species of Penicillium and Aspergillus fungi, is often detected in apple juices and ciders. Previous research has shown the effectiveness of granular activated carbon for reducing patulin levels in aqueous solutions, apple juices, and ciders. In this study, ultrafine activated carbon was bonded onto granular quartz to produce a composite carbon adsorbent (CCA) with a high carbonaceous surface area, good bed porosity, and increased bulk density. CCA in fixed-bed adsorption columns was evaluated for efficacy in reducing patulin levels from aqueous solutions and apple juice. Columns containing 1.0, 0.5, and 0.25 g of CCA were continuously loaded with a patulin solution (10 microg/ml) and eluted at a flow rate of 1 ml/min. Results indicated that 50% breakthrough capacities for patulin on 1.0-, 0.5-, and 0.25-g CCA columns were 137.5, 38.5, and 19.9 microg, respectively. The effectiveness of CCA to adsorb patulin and prevent toxic effects was confirmed in vitro using adult hydra in culture. Hydra were sensitive to the effects of patulin, with a minimal affective concentration equal to 0.7 microg/ml; CCA adsorption prevented patulin toxicity until 76% breakthrough capacity was achieved. Fixed-bed adsorption with 1.0 g of CCA was also effective in reducing patulin concentrations (20 microg/liter) in a naturally contaminated apple juice, and breakthrough capacities were shown to increase with temperature. Additionally, CCA offered a higher initial breakthrough capacity than pelleted activated carbon when compared in parallel experiments. This study suggests that CCA used in fixed-bed adsorption systems effectively reduced patulin levels in both aqueous solutions and naturally contaminated apple juice; however, the appearance and taste of apple juice may be affected by the treatment process.

Investigation of Organophilic Montmorillonite Clay Inclusion in Zearalenone-Contaminated Diets Using the Mouse Uterine Weight Bioassay

Previous studies with low-pH montmorillonite (LPHM) clay exchanged with alkylammonium compounds showed that these organo clays were quite effective in sorbing the estrogenic mycotoxin zearalenone (ZEN) from aqueous solution. The potential toxicity of these types of clays, in particular hexadecyltrimethylammonium (HDTMA) LPHM, led to the investigation of the sorption efficacy of clay exchanged with a less toxic primary amine analog, hexadecylamine (HDA). Isothermal analysis studies showed that HDA LPHM was able to bind ZEN, but less effectively than HDTMA LPHM as evidenced by a significantly lower Freundlich K (63,900 vs. 845). The in vivo effectiveness of these two clays to bind ZEN was tested utilizing the mouse uterine weight bioassay. At a dietary inclusion level of 0.25%, the clays did not have a negative impact on overall animal health as measured by final body weight; however, they did not protect the animals from the estrogenic effects induced by 35 mg ZEN/kg in the feed (i.e., the uterine weights were not reduced in comparison to ZEN alone). In fact, the HDTMA LPHM group showed an increase in uterine weight that was more than the ZEN treatment group. When the animals were fed 0.5% clay, both exchanged clays (i.e., HDTMA LPHM and HDA LPHM) resulted in decreased body weight gain. The uterine weights of ZEN-fed animals (either alone or in combination with clays) were not significantly different from each other. In contrast, the uterine:body weight ratio showed a dramatic increase in the groups fed exchanged clay + ZEN compared to ZEN alone. These results suggest that alkylamine groups may assist the transport or uptake of ZEN and result in an enhanced toxicity from contaminated feed. The findings from this study clearly demonstrate the need for careful testing of all mycotoxin-binding agents before their inclusion in the diet.

Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay

The ability of ozone gas (O3) to detoxify zearalenone (ZEN), a commonly occurring estrogenic mycotoxin, was assessed utilizing the mouse uterine weight bioassay. Solutions containing 12 ppm ZEN in water were ozonated for varying time periods (0, 0.5, and 5 min), then extracted with chloroform and evaporated to dryness. The residue was redissolved in acetonitrile and analyzed for ZEN. High-performance liquid chromatography (HPLC) analysis of aliquots indicated a rapid degradation and decline in parent ZEN level with increasing time of ozone treatment. The acetonitrile solution containing the degraded ZEN residue was added to a known volume of corn oil and evaporated under nitrogen to eliminate the acetonitrile in the oil. Eighteen-day-old prepubertal female mice (B6C3F1 strain) were gavaged daily with the test chemicals in 50 microl of corn oil between d 18 and 23. Initial dose-response studies showed that a concentration of 60 microg ZEN/mouse/d produced uterine weights that were significantly higher than the uterine weights of control animals (2.7 times higher than that of the solvent control). Treatment groups for the ozonation study included: DES, 0.1 microg (positive control), untreated ZEN (60 microg), extraction control for ZEN (60 microg), 0.5 min ozone-treated ZEN (60 microg), 5 min ozone-treated ZEN (60 microg), solvent control (50 microl), and absolute control. Results showed the uterine weights of animals receiving the ozone-treated ZEN were not significantly affected. These findings were in agreement with HPLC analyses and suggested that ozone can prevent the estrogenic effects of this important mycotoxin in mice. Importantly, ozone treatment of contaminated whole grains may enable the practical detoxification and control of ZEN. Also, the mouse uterine weight bioassay may be useful in assessing the efficacy of other detoxification strategies for estrogenic chemicals.

Molecular characterization of high affinity, high capacity clays for the equilibrium sorption of ergotamine

Ergot alkaloids (mycotoxins) produced by Claviceps and Neotyphodium species of fungi may contaminate animal feedstuffs and results in disease in livestock. In this study, diverse phyllosilicate clays and other adsorbent materials, differing in chemical and structural characteristics, were tested for their ability to sorb ergotamine, a prevalent ergot mycotoxin, from acidic solution. Results indicated minimal binding to those sorbents possessing low surface area, cation exchange capacity and inaccessible interlayer regions. Cetyl pyridinium-exchanged montmorillonite (organoclay) exhibited decreased propensity for ergotamine in acidic solution as compared with the unexchanged hydrophilic parent clay. The highest ergotamine sorption was observed with cation exchanged montmorillonite clays; whereas, when collapsed, these same clays sorbed very little ligand. Based on initial binding experiments, calcium and sodium montmorillonite clays were prioritized for further characterization, including: capacity, affinity, and heat (enthalpy) of adsorption. Computer models of energy-minimized ergotamine isomers and clay were used to illustrate possible mechanisms of ergot alkaloid sorption at interlayer sites. Additional studies are warranted to assess the stability of ergot alkaloid/clay complexes under alkaline conditions to further understand the mechanism of adsorption.

Utilization of electrochemically generated ozone in the degradation and detoxication of benzo[a]pyrene

The ability of electrochemically generated ozone (O3) to degrade and detoxify the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) was assessed utilizing the chick embryotoxicity screening test (CHEST) and Hydra attenuata bioassays. Aqueous solutions containing 10 microg/ml BaP and 0.5% (v/v) acetonitrile were subjected to ozonolysis for 0 to 30 min. Rapid degradation of BaP was evident by both gas chromatography/mass spectroscopy (GC/MS) and high-performance liquid chromatography (HPLC) analysis. HPLC fluorescence detection revealed no BaP shortly after 5 min of ozonolysis, while HPLC with PDA detection demonstrated continued reactions with ozone over the 30-min time course. As little as 2 min of O3 treatment afforded protection from BaP-induced mortality and toxicity (embryolethality and liver discoloration) in the chicken embryos. In the hydra bioassay, no toxicity was observed in the adult hydra until the ozonolysis products were reconstituted 100-fold from their initial post-ozonolysis concentrations. The results obtained from this study clearly demonstrate the potential application of electrochemically generated O3 for the detoxication and prevention of toxicity of BaP. Both CHEST and hydra assays predict that the ozonolysis products of BaP are less toxic than the parent compound.