Effects of duration of treatment and dosage of eicosapentaenoic acid and stearidonic acid on red blood cell eicosapentaenoic acid content.

OBJECTIVE: The purpose of this randomized, controlled, parallel group study was to characterize the relationships between dosages of stearidonic acid (SDA) and eicosapentaenoic acid (EPA), and incorporation of EPA into red blood cell (RBC) membranes over time. METHODS: Healthy subjects (n=131) received capsules with placebo (safflower oil), SDA (0.43, 1.3, 2.6, or 5.2 g/d) or EPA (0.44, 1.3, or 2.7 g/d) for 12 weeks. RBC fatty acids were analyzed biweekly. RESULTS: RBC %EPA increased in all EPA and SDA groups (p<0.02 vs. control) except the 0.43 g/d SDA group (p=0.187). For theoretical intakes of EPA of 0.25, 0.5, and 0.89 g/d, the amounts of SDA needed to achieve equivalent RBC EPA enrichment were 0.61, 1.89, and 5.32 g/d (conversion efficiencies of 41%, 26%, and 17%), respectively. CONCLUSIONS: SDA increased RBC %EPA in a dosage and time-dependent manner at intakes as low as 1.3 g/d.

Evaluation of methods for predicting the toxicity of polycyclic aromatic hydrocarbon mixtures.

Risk assessments of polycyclic aromatic hydrocarbon mixtures are hindered by a lack of reliable information on the potency of both mixtures and their individual components. This paper examines methods for approximating the toxicity of polycyclic aromatic hydrocarbon (PAH) mixtures. PAHs were isolated from a coal tar and then separated by ring number using HPLC. Five fractions (A-E) were generated, each possessing a unique composition and expected potency. The toxicity of each fraction was measured in the Salmonella/mutagenicity assay and the Chick Embryo Screening Test (CHEST). Their abilities to induce ethoxyresorufin-O-deethylase and to inhibit gap junction intercellular communication in rat liver Clone 9 cells were also measured. In the Salmonella/mutagenicity assay, fractions were predicted to have potencies in the order C > D > E > B > A. Toxic equivalency factors (TEFs) for fractions A-E were in the order E > or = D > C > B > A. TEF values were 20,652, 20,929, 441, 306, and 74.1 micrograms of BaP equiv/g, respectively. A lack of agreement between assay-predicted potencies and chemical analysis-predicted potencies was observed with other assays and other methods of calculation. The results demonstrate the limitations of using a single method to predict the toxicity of a complex PAH mixture.

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.

Deamination of fumonisin B(1) and biological assessment of reaction product toxicity.

Fumonisin B(1), a potent mycotoxin found in grain, has been resistant to degradation and detoxification by a variety of methods, including milling, fermentation, ammoniation, and ozonation. The primary amine of this compound contributes significantly to its toxicity; therefore, the major aim of this research was to remove this moiety via diazotization. In this study, fumonisin B(1) was deaminated in aqueous solution under conditions of acidic pH and low temperature (pH 1.0 and 5 degrees C) with the addition of NaNO(2). The concentration of fumonisin B(1) in the solution was analyzed by HPLC using o-phthaldialdehyde to derivatize the primary amine. Progress of the reaction was monitored as a loss of the derivatized peak as observed by HPLC with fluorescence detection. TLC analysis showed the disappearance of fumonisin B(1) following diazotization. Further, TLC displayed at least four reaction products that were not primary amines. Matrix-assisted laser desorption/ionization mass spectrometry coupled with time-of-flight analysis of the diazotization products also showed a diminished amount of authentic fumonisin B(1) and allowed identification of a product formed by the replacement of the primary amine with a hydroxyl group. The adult Hydra attenuata bioassay indicated a marked decrease in the toxicity of the products in comparison to parent fumonisin B(1). Optimization of this reaction could result in a rapid and practical method for the reclamation of fumonisin B(1)-contaminated feeds.

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.

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.

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.