Kittane Mayura

OXIDATIVE DEGRADATION AND DETOXIFICATION OF MYCOTOXINS USING A NOVEL SOURCE OF OZONE

Practical methods to degrade mycotoxins using ozone gas (O3) have been limited due to low O3 production capabilities of conventional systems and their associated costs. Recent advances in electrochemistry (i.e. proton-exchange membrane and electrolysis technologies) have made available a novel and continuous source of O3 gas up to 20% by weight. It is possible that the rapid delivery of high concentrations of O3 will result in mycotoxin degradation in contaminated grains--with minimal destruction of nutrients. The major objectives of this study were to investigate the degradation and detoxification of common mycotoxins in the presence of high concentrations of O3. In this study, aqueous equimolar (32 microM) solutions of aflatoxins B1 (AfB1), B2 (AfB2), G1 (AfG1), G2 (AfG2), cyclopiazonic acid (CPA), fumonisin B1 (FB1), ochratoxin A (OA), patulin, secalonic acid D (SAD) and zearalenone (ZEN) were treated with 2, 10 and/or 20 weight% O3 over a period of 5.0 min and analysed by HPLC. Results indicated that AfB1 and AfG1 were rapidly degraded using 2% O3, while AfB2 and AfG2 were more resistant to oxidation and required higher levels of O3 (20%) for rapid degradation. In other studies, patulin, CPA, OA, SAD and ZEN were degraded at 15 sec, with no by-products detectable by HPLC. Additionally, the toxicity of these compounds (measured by a mycotoxin-sensitive bioassay) was significantly decreased following treatment with O3 for 15 sec. In another study, FB1 (following reaction with O3) was rapidly degraded at 15 sec, with the formation of new products. One of these appeared to be a 3-keto derivative of FB1. Importantly, degradation of FB1 did not correlate with detoxification, since FB1 solutions treated with O3 were still positive in two bioassay systems.

Aroclor 1254 as an antagonist of the teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 20 micrograms/kg) to pregnant C57BL/6J mice (on day 10) resulted in 62% fetuses with cleft palate per litter without any observable maternal toxicity. In contrast, Aroclor 1254 administered at a dose of 750 mumol/kg was not teratogenic. Cotreatment of the pregnant mice with both Aroclor 1254 (244 mg/kg) and 2,3,7,8-TCDD (20 micrograms/kg) resulted in an 8.2% incidence of cleft palate per litter. In contrast, Aroclor 1254 did not afford any protection from the teratogenicity of dexamethasone in C57BL/6J mice. Previous studies have shown that Aroclor 1254 can act as a partial antagonist of the microsomal enzyme induction and immunotoxic effects of 2,3,7,8-TCDD in C57BL/6J mice and this paper demonstrates that the commercial polychlorinated biphenyl mixture also antagonizes 2,3,7,8-TCDD-mediated teratogenicity in this strain of mice.

DEVELOPMENT OF POROUS CLAY-BASED COMPOSITES FOR THE SORPTION OF LEAD FROM WATER

Lead contamination of water is a major health hazard, as illustrated by the fact that exposure to this metal has been associated with death and disease in humans, birds, and animals. The present research was aimed at the development of a porous, solid-phase sorbent that can be used in the remediation of lead-contaminated water. A suitable sorbent was identified by screening various clays and other materials for their ability to effectively bind lead. The clay was adhered to a solid support using an aqueous solution of carboxymethyl cellulose. The binary composite was then tested for its ability to bind lead from solution, while providing void volume, increased surface area, and considerably enhanced hydraulic conductivity. The results suggested that a combination of sodium montmorillonite clay and carbon exhibited enhanced sorption of lead compared to carbon alone, and also supported the potential application of various combinations of sorbent materials. This value-added combination of clay, solid support, and adhesive will allow for the construction of column filtration systems that are multifunctional and capable of purifying large volumes of contaminated water.

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.

Effects of phyllosilicate clay on the metabolic profile of aflatoxin B1 in Fischer-344 rats

The phyllosilicate clay, hydrated sodium calcium aluminosilicate (HSCAS), has been shown to prevent aflatoxicosis in farm animals by reducing the bioavailability of aflatoxin. The present study was designed to determine the effects of HSCAS on the metabolism of aflatoxin B1 (AFB1) in an aflatoxin-sensitive species. Male Fischer-344 rats were orally dosed with 1.0, 0.5, 0.25 and 0.125 mg AFB1/kg body weight alone and in combination with 0.5% HSCAS. Urine samples were collected after 6, 24, 36, and 48 h. Aflatoxin M1 (AFM1) and aflatoxin P1 (AFP1) were detected in most urine samples, with or without HSCAS. AFM1 was found to be the major metabolite. Metabolite concentrations were significantly decreased in the presence of HSCAS, and more importantly, no additional metabolites were detected. Our results suggest that the AFB1-HSCAS complex was not significantly dissociated in vivo, and support earlier findings that HSCAS tightly binds aflatoxin.

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.

Effect of simultaneous prenatal exposure to ochratoxin A and citrinin in the rat

Ochratoxin A (OA) and citrinin (CT) are food-borne mycotoxins produced by several fungal species of the genera Aspergillus and Penicillium. Both are teratogenic in the rat. To determine the prenatal effects of simultaneous exposure to these toxins, pregnant Sprague-Dawley rats were injected either with a single individual subthreshold teratogenic dose of OA (1 mg/kg) or CT (30 mg/kg) or with both toxins. Toxins were dissolved in 5% sodium bicarbonate and administered subcutaneously on one of gestation d 5, 6, 7, 8, 10, 11, or 14. Maternal body weight gain of animals in the combination group was similar to other treatment groups and the control. Approximately 22-40% mortality in dams occurred on gestation d 5, 6, 7, and 14. Other than d 7, there was no significant effect on the number of implants. Treatment on d 5 or 7 resulted in increased fetal resorptions. Fetal body weights were not decreased significantly. OA and CT in combination resulted in a significant increase in gross malformations on d 6 and 7, visceral anomalies on d 5, 7, 8, and 10, and skeletal defects on d 5, 6, 7, 8, 10, and 14. When administered individually, OA and CT resulted in very few fetal resorptions. Fetal body weights were not significantly different except on d 8 of gestation following CT treatment. Individual toxin treatment resulted in minimal malformations on all gestation days. These results suggest that OA and CT, when administered concurrently, may interact to enhance prenatal toxicity and teratogenicity, and these results have focussed attention on the public health hazards of contamination of food with these mycotoxins.

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.

Evaluation of the developmental toxicity of chlorinated phenols utilizing Hydra attenuata and postimplantation rat embryos in culture.

Chlorinated phenols (CPs) represent a major component of hazardous oily and wood-preserving wastes that are widely distributed in chemical dumpsites throughout the United States. Pentachlorophenol (C5P) has been reported to be highly embryolethal and embryotoxic in rats. However, data pertaining to the developmental toxicities of other important CPs are limited. In this study, the toxicities of phenol, CP homologues and their isomers, selected phenyl acetates, anisoles, sodium phenates, and tetrachlorobenzoquinones (a total of 38 chemicals) were evaluated using cultures of Hydra attenuata (HA). Developmental hazard index (A/D ratio) was determined for selected test chemicals (i.e., those chemicals which resulted in an early toxic endpoint at the lowest whole-log concentration in the adult hydra assay). These same chemicals were evaluated at equimolar concentration in postimplantation rat whole embryo culture (WEC). HA and WEC studies demonstrated a linear relationship between toxicity and the degree of chlorine substitution with C5P greater than 2,3,4,5-C4P greater than 2,3,5-C3P greater than 3,5-C2P greater than 4-CP greater than phenol. The A/D ratios from the HA assay were approximately 1 for all of the chemicals tested. Findings from the WEC assay indicated similar results based on growth, gross morphology, and DNA and protein content of embryos. The results obtained in the HA and WEC assays suggest that the chlorinated phenols are not potent teratogens. The combination of HA and WEC may facilitate the rapid detection and ranking of hazardous chemicals associated with complex mixtures of chemical wastes.

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.