Confirmation of reduced toxicity of deoxynivalenol in extrusion-processed corn grits by the MTT bioassay

The objective of this study was to determine the loss of toxicity of deoxynivalenol in extruded cereal-based products by the tetrazolium salt (MTT) bioassay using a sensitive Chinese hamster ovary (CHO-K1) cell line and to compare the results to chemical (high-performance liquid chromatography, HPLC) and biochemical (enzyme-linked immunosorbant assay, ELISA) methods of analysis. A split-split plot design was used for the extrusion process experiments at temperatures of 150, 175, and 200 degrees C and screw speeds of 70 and 140 rpm. The initial mean deoxynivalenol concentration in the corn grits artificially contaminated with Fusarium graminearum was found to be 23.5 mug/g as measured by HPLC. The percent reductions of deoxynivalenol in the contaminated corn grits upon extrusion processing ranged from 22 to 35%, from 21 to 34%, and from 21 to 37% as measured by HPLC, ELISA, and MTT bioassay, respectively. The MTT bioassay results were more closely correlated with HPLC (r = 0.90) results than with ELISA results (r = 0.78). The MTT bioassay, using a sensitive mammalian cell line, was demonstrated to be a useful method for quantification of deoxynivalenol as well as a potential toxicity screening method for contaminated extruded cereal-based products.     

Determination of cytotoxic trichothecenes in corn by cell culture toxicity assay

The great sensitivity of some cell species to toxins has been adapted to a direct biological determination of trichothecene contamination of food and feeds. The murine spleen lymphocyte stimulated by PHA (Phaseolus vulgaris phytohaemagglutinin) appeared to be the most convenient cells because of their particular sensitivity to cytotoxic trichothecenes and the opportunity to translate this cytotoxicity to immunosuppressive hazard, one of the most important concerns for trichothecenes. In this paper, the use of cell cultures was adapted for a survey of corn. The toxins were extracted by aqueous methanol, and the extract was defatted with hexane and purified on a silica gel/Florisil column. This extract was then used for a gas chromatographic (GC) determination and the biological test. The growth of cells was measured by the incorporation of tritiated thymidine (3H Tdr), and the inhibition was expressed by the IC50: concentration of corn extract inhibiting by half the 3H Tdr incorporation. We have tested pure toxins, control corn, corn spiked with T-2 toxin, corn experimentally inoculated with toxigenic Fusarium strains, and naturally contaminated corn. A good correlation exists between IC50 and the T-2 toxin concentration as determined by GC analysis. The response is not affected by the presence of zearalenone or by small amounts of deoxynivalenol. A quantitative evaluation of cytotoxic trichothecenes in corn is valuable in the range of 100 ppb to 10 ppm, expressed as T-2 toxin equivalents. The result is obtained in 48 h.     

Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with Zebrafish embryos

Goals, Scope and Background  While water quality strongly improved over decades in the Rhine River, sediments still reflect elapsed contaminations of organic pollutants and heavy metals. In comparing genotoxic effects induced by both sediment extracts and whole sediments, a ratio of bioavailable toxicity and total extractable toxicity is obtained. Since contaminated sites whose contaminants are toxic and as well bioavailable present an elevated risk to the ecosystem, such ratios may be used as a warning signal to identify sites of primary concern. Methods  Accordingly, two different exposure scenarios were compared to reveal the genotoxic potential of 18 sediment samples derived from 9 sample sites along the River Rhine. For assessment of effects on genome integrity, DNA fragmentation was measured using the comet assay with primary cells isolated from zebrafish embryos previously exposed to either organic sediment extracts or freeze-dried sediments at sublethal concentrations. Additionally, chemical data were used to determine responsible pollutants and correlate them with biological effects. Results  Whereas 17 out of 18 sediment extracts caused significant DNA damage to the embryo cells, only 4 native sediments showed a genotoxic potential. Thus, under field-like exposure conditions, a major part of potentially genotoxic compounds seem to remain particle-bound and ineffective, as shown for whole sediment exposure. Conversely, the organic extracts seem to contain enriched concentrations even of hardly soluble substances. Hence, organic extracts may be used as a screening tool to address potentially polluted sites, even though the relevance of these results for the field situation may be questionable. Investigations on native sediments determined few sites with bioavailable and therefore ecologically most relevant genotoxic sediment compounds. Discussion  However, these results may underestimate the total hazard potential of sample sites with hardly bioavailable substances. Chemical data revealed a variety of anthropogenic pollutants, ranging from PAHs to heavy metals. Nevertheless, chemical data on the measured priority pollutants did not fully explain the pollution pattern of the bioassays but clearly determined substances of concern (e.g., HCB, heavy metals) in particular sample sites. Conclusions  There is a striking advantage in assessing the genotoxicity by means of different exposure scenarios that focus on either bioavailable or extractable fractions, as the combination of the results allows obtaining information on specific properties of the genotoxicants and their bioavailability. An additional correlation with chemical data should be required to identify priority pollutants, as long as the responsible contaminant is known a priori. As many studies revealed inherent failures of such a correlation, an effect-driven analysis of pollutants is recommended as a promising tool to identify even non-priority pollutants by means of their ecotoxicological effectiveness.     

Effect of industrial processing on the distribution of aflatoxins and zearalenone in corn-milling fractions

The aim of this study was to investigate the distribution of aflatoxins and zearalenone levels in various corn-milling fractions. Corn kernels and six derived milling fractions (germ, bran, large and small grits, flour, and animal feed flour) were sampled in an industrial plant; both conventional and organic corns were sampled. To evaluate the effect of cooking, samples of polenta were prepared starting from naturally contaminated flour. Conventional and organic lots showed mycotoxin contamination. For both lots, germ, bran, and animal feed flour showed a marked concentration factor from 239 to 911% accounting for both the low yields of the derived products and the distribution of aflatoxins and zearalenone contamination in the outer parts of the kernels. Conversely, a reduction factor of at least four times from raw material to finished products was observed. Polenta samples were unaffected by the cooking process, with levels of contamination similar to those of starting flour.     

Evaluation of Monier-Williams and Committee methods for bisulfite determination as used by the potato processing industry

The Monier-Williams and the iodometric Committee methods were evaluated for the determination of bisulfite in potato products. Both methods showed critical problems in accuracy and precision at concentrations below 60 ppm. Sensitivity levels of 25 ppm are possible with the Monier-Williams method. However, accuracy problems reflected by the coefficients of variation (Monier-Williams, 47.4%; Committee, 78.2%) as well as variability in recoveries and blank values for both methods produce qualitative rather than quantitative analytical data at low SO2 concentrations.     

Effect of processing of black currant press-residue on polyphenol composition and cell proliferation

The press-residue of black currants provides a good source of phenolic antioxidants. The purpose of this study was to optimize the extraction of phenolic compounds from the press-residue by use of extraction conditions compatible with food use. The effects of temperature, extraction duration, and use of ultrasound-assisted extraction on the juice yield, total phenolics (TP), and anthocyanin content of aqueous extracts were studied. Within the variables and response factors tested, the optimal conditions were a 15 min extraction at 90 °C. No significant effect from ultrasound-assisted extraction was found. The composition of anthocyanins and polyphenols was highly dependent on the extraction temperature. The percentage contribution of delphinidin- and cyanidin-3-rutinoside to TP had a negative linear correlation with temperature, while delphinidin- and cyanidin-3-glucoside had a positive linear correlation with temperature, with a maximum amount obtained at 80 °C and 55 °C, respectively. Furthermore, extracts obtained at higher temperatures showed a stronger inhibition of proliferation of Caco-2, HT-29, and HCT 116 cells than extracts obtained at lower temperatures. This may be due to the decomposition of complex polyphenols at higher temperatures, making them more accessible to the cells.     

Curcuminoids-cellular uptake by human primary colon cancer cells as quantitated by a sensitive HPLC assay and its relation with the inhibition of proliferation and apoptosis

Curcumin, which is a bright orange-yellow pigment of turmeric with antioxidant properties, has been shown to produce a potent preventative action against several types of cancers in recent studies. It has also been reported to protect the development of colon tumor in animals being fed with carcinogen. In the colon cancer cells, curcumin was illustrated to inhibit cell proliferation and induce apoptosis. As an antioxidant, it acts as an anti-inflammatory as well as an antitumor agent. Curcumin has been detected to exist in nature in the form of curcuminoids, a mixture of curcumin, the major component, with two of its related demethoxy compounds (demethoxycurcumin and bisdemethoxycurcumin). In the present study, we have investigated the antiproliferation and induced apoptosis effects of curcuminoids on colon cancer, using the primary cancer cells isolated from Taiwanese colon cancer patients as the model for colorectal cancer. Results showed that curcuminoids inhibited cell proliferation and induced apoptosis of these human primary colon cancer cells. The effects were observed in a dose-dependent manner as dose increased from 12.5 to 100 microM. With the aim of furthering the fundamental understanding of the mechanisms underlying the antiproliferation and induced apoptosis effects of curcuminoids on these human colon cancer cells, we developed a sensitive, rapid, and reproducible assay method based on high-performance liquid chromatography (HPLC). This HPLC technique developed was found to successfully determine, in a quantitative manner, the cellular uptake of curcuminoids. The uptake of these curcuminoids by the colon cancer cells was shown to increase as the dose of curcuminoids was increased. The observations of inhibited proliferation and increased apoptosis in the colon cancer cells appeared to be associated with the cellular uptake of curcuminoids.     

Long distance transport of anthropogenic lead as measured by lake sediments

Long-term atmospheric deposition of Pb has been measured using lake sediment cores. The average ‘whole-lake’ Pb burden was measured for several lakes in each of four regions located throughout Ontario and Quebec, Canada. Since the regions were remote to any point source of Ph, large scale variation in atmospheric transport could be studied. The results indicate that Pb deposition is highest in South-Central Ontario, where lake burdens ranged from 312 to 432 mg m^?2, and that it decreases in an eastward direction into Quebec. Lowest deposition was at the site farthest north, Schefferville, Quebec, where fallout was 31 to 42 mg m^?2, less than one-tenth of that previously observed in South-Central Ontario. Lead-210 dating of sediment cores from the northern site showed that input to these remote lakes has occurred over the same time period as in the southern sites.     

Quantification of zearalenone in various solid agroenvironmental samples using D6-zearalenone as the internal standard

Because of its pronounced estrogenicity, zearalenone may be of concern not only in the aqueous but also in the terrestrial environment. Therefore, we developed several analytical methods to quantify zearalenone in different solid matrices of agroenvironmental relevance (i.e., plant organs, soil, manure, and sewage sludge). The use of D(6)-zearalenone as the internal standard (IS) was essential to render the analytical method largely matrix-independent because it compensated for target analyte losses during extract treatment and ion suppression during ionization. Soil and sewage sludge samples were extracted with Soxhlet, whereas plant material and manure samples were extracted by liquid solvent extraction at room temperature. Absolute recoveries for zearalenone were 70-104% for plant materials, 105% for soil, 76% for manure, and 30% for sewage sludge. Relative recoveries ranged from 86 to 113% for all matrices, indicating that the IS was capable to largely compensate for losses during analysis. Ion suppression, between 8 and 74%, was in all cases compensated by the IS but influenced the method quantification levels. These were 3.2-26.2 ng/g(dryweightdw) for plant materials, 0.7 ng/g(dw) for soil, 12.3 ng/g(dw) for manure, and 6.8 ng/g(dw) for sewage sludge. Plant material concentrations varied from 86 ng/g(dw) to more than 16.7 microg/g(dw), depending on the organ and crop. Soil concentrations were between not detectable and 7.5 ng/g(dw), depending on the sampling depth. Zearalenone could be quantified in all manure samples in concentrations between 8 and 333 ng/g(dw). Except for two of the 85 investigated sewage sludge samples, zearalenone concentrations were below quantification limit.     

Elemental composition of the rice plant as affected by iron toxicity under field conditions

Abstract

Iron (Fe) toxicity is a major nutrient disorder affecting the production of wetland rice in the humid zone of West Africa. Little attention has been given to determining the macro‐ and micronutrient composition of rice plants grown on wetland soils where Fe toxicity is present although results from such study could provide useful information about the involvement of other nutrients in the occurrence of Fe toxicity. A field experiment was conducted in the 1997 dry season (January‐May) at an Fe toxic site in Korhogo, Ivory Coast, to determine the elemental composition of Fe tolerant (CK 4) and susceptible (Bouake 189) lowland rice varieties without and with application of nitrogen (N), phosphorus (P), potassium (K), and zinc (Zn). For both Fe‐tolerant and susceptible varieties, there were no differences in elemental composition of the whole plant rice tops, sampled at 30 and 60 days after transplanting rice seedlings, except for Fe. All the other nutrient element concentrations were adequate. Both Fe‐tolerant and susceptible cultivars had a high Fe content, well above the critical limit (300 mg Fe kg^‐1 plant dry wt). These results along with our observations on the elemental composition of rice plant samples collected from several wetland swamp soils with Fe toxicity in West Africa suggest that “real”; iron toxicity is a single nutrient (Fe) toxicity and not a multiple nutrient deficiency stress.     

In vivo staining of reduced iron by 2,2′ bipyridine in rice exposed to iron toxicity

A protocol for a novel method to visualize Fe(II) in rice tissues is proposed. The method is based on the selective formation of a purple‐red color complex of 2,2′ bipyridine and Fe(II). Rice genotypes were exposed to 18 mM Fe(II) in nutrient solution for 2 d. Root systems of intact plants were subsequently placed in 2,2′ bipyridine solutions. The formation of the [Fe(bipy)<$>_3^{2+}<$>] color complex was visualized using bifocal microscopy. The method may improve the selection of genotypes during breeding for Fe‐toxicity resistance of rice.     

Temporal variation in the hydraulic conductivity of a tilled clay soil as measured by tension infiltrometers

Steady-state infiltration rates from tension infiltrometers were measured on ploughed and unploughed plots in a clay soil during the period June to October. Measurements were made both at the soil surface and at depths of 15 and 25 cm. Hydraulic conductivity in the water potential range zero to ?11 cm was obtained using a piece-wise exponential K(Ψ) function and Wooding's solution for infiltration from a circular source. A two-line regression model showed excellent fits to paired (In KΨ) values on all measurement occasions. This may indicate the existence of a bimodal pore system, reflecting the contributions of macro- and mesopores to the measured K(Ψ) function. The break-point potential dividing the two pore systems varied between c.?4 and ?6cm. Significant variations in the K(Ψ) function between sampling occasions were found at the soil surface, but not at depths of 15 and 25 cm. Measured K(Ψ) values decreased during the growing season, particularly at potentials between ?4 and ?6 cm where reductions were up to one order of magnitude. This was attributed to soil structural breakdown by rain impact and surface capping or sealing. Hydraulic conductivity near the soil surface was significantly increased by disc harrowing in autumn. In contrast, no pronounced difference in the K(Ψ) function between ploughed and unploughed treatments could be discerned at 15 and 25 cm depths in the soil.     

Identification of an aliphatic epoxide and the corresponding dihydrodiol as novel congeners of zearalenone in cultures of Fusarium graminearum

The mycotoxin zearalenone (ZEN) is produced by various Fusarium fungi and frequently found as a contaminant in food and feed. There are reports in the literature that several closely related analogues of ZEN are also formed in cultures of Fusarium species. We have therefore analyzed the organic extract from a 40 day culture of Fusarium graminearum by LC-DAD-MS and detected 15 compounds, which could be congeners of ZEN because of their ultraviolet, mass spectroscopy, and tandem mass spectroscopy spectra. In addition to confirming the previously reported α- and β-stereoisomers of 5-hydroxy-ZEN and 10-hydroxy-ZEN, we identified seven ZEN congeners for the first time. One of the major novel congeners was shown by nuclear magnetic resonance spectroscopy and chemical synthesis to have the structure of an aliphatic ZEN epoxide, whereas two minor products proved to be the corresponding dihydrodiols. In addition, three stereoisomers of a cyclization product of the dihydrodiols, carrying a spiro-acetal group, were identified as fungal products for the first time. The latter may be artifacts, because the ZEN epoxide and dihydrodiol are unstable under acidic conditions and rearrange easily to the spiro-acetal compounds.     

Manganese toxicity in bush bean as affected by concentration of manganese and iron in the nutrient solution

An experiment was conducted to clarify the relationship between Mn toxicity and Fe deficiency in bush snap bean (Phaseolus vulgaris L. cv. ‘Wonder Crop No. 2'). Seedlings were grown in full strength Hoagland No. 2 solution at pH 6.0 for ten days. Six concentrations of Mn as MnCl₂.4H₂O were used in combination with three concentrations of Fe as FeEDTA.

Toxicity symptoms, induced by low levels of Mn (0.1 ppm and above), included: small brown necrotic spots and veinal necrosis on primary leaves; necrosis on primary leaf petioles; interveinal chlorosis, with or without brown necrotic spots, on trifoliate leaves; and brown necrotic spots on stipules. Manganese toxicity symptoms were alleviated or prevented by increasing Fe concentration in the nutrient solution.

Manganese concentration in the leaves increased with increasing Mn and decreased with increasing Fe concentration in the nutrient solution, Iron concentration in the roots increased with increasing Fe concentration in the nutrient solution; however, Fe concentration in the leaves was not significantly affected by increasing Mn concentration in the solution culture. Manganese toxicity symptoms developed when Mn concentration in the leaves reached about 120 ppm.

A decrease in the Fe/Mn ratio in the nutrient solution resulted in a proportionate decrease in that of the leaves. Manganese toxicity symptoms occurred when the Fe/Mn ratio in the solution was 10.0 and below, or when the ratio in the leaves was less than 1.5. The ratio of Fe/Mn in the solution required for optimum growth of ‘Wonder Crop No. 2’ bean, without Mn toxicity symptoms, was in the range of 20.0 to 25.0.

Results indicate that the chlorosis on bush bean leaves induced by excessive Mn in the nutrient solution was due to excessive accumulation of Mn and not to Fe deficiency.     

Effects of land use and clay content on soil structure as measured by Fracture Surface Analysis

Soil clods were collected from the A horizons of six soils (3 Aqualfs and 3 Uderts) in the vicinity of Bayreuth in northern Bavaria. The soils had a range of clay contents and came from arable, meadow (pasture) and forest areas. The soils ranged in workability from easily workable to difficult to work. The clods, still moist as collected, were fractured into two parts by tensile stress and one part of each clod was embedded in polyester resin. The embedded clod parts were then sectioned to show profiles of the fracture surfaces. The profiles were digitized by a television scanner. Statistical measures of the roughness of the fracture surfaces were computed. Fracture surface roughness was strongly influenced by land use and soil clay content. Soil workability was related to fracture surface roughness and hence also to land use and soil clay content. Ease of soil working was mainly associated with the presence of soil structural features larger than 10 mm.     

The effect of starvation on the planarian Arthurdendyus triangulatus (Tricladida: Terricola) as measured by stable isotopes

Many studies of the population dynamics of the planarian Arthurdendyus triangulatus have been confounded because it has not been possible to define if small flatworms are adults that have been starved or young ones being recruited into the population. Body shrinkage is a process exhibited by planarians under nutritional stress. In birds and the few invertebrate species studied, starvation leads to a ¹⁵N-enrichment of body tissues (i.e. an increased δ ¹⁵N). Under laboratory conditions, the posterior parts of flatworms were removed at day 0 and frozen and compared isotopically with the anterior parts of flatworms, which were starved for 67, 108, 171 and 243 days. With one exception, δ ¹³C 108 days after starvation, the impact of starvation on A. triangulatus %N, %C and δ ¹³C was not significant statistically. In contrast, δ ¹⁵N increased significantly (to a maximum enrichment of 2.8‰ by day 243) on all sampling dates. This is supported by significant positive correlations between the changes in δ ¹⁵N between the anterior and posterior body parts and number of days after starvation (p≤0.05) and decrease in body weight (p≤0.01), respectively. Existing δ ¹⁵N data from field populations of animals show intra-population variability of around 5.0‰, greater than that noted as a result of starvation. Thus, as a single measure, it is unlikely that δ ¹⁵N can be used to accurately characterise the proportion of starved A. triangulatus within a field population.     

Na+,K+-ATPase调节肝再生增强因子促HepG2细胞增殖

采用四甲基噻唑盐(M TT)法检验肝再生增强因子(ALR)对H epG 2细胞增殖作用;[3H]-T dR掺入测定细胞DNA合成;采用无机磷比色法测定细胞N a ,K -ATPase的酶活力。结果表明:ALR通过促进H epG 2细胞DNA合成,使细胞增殖,并存在剂量效应正相关性(P<0.01);ALR对N a ,K -ATPase酶活呈剂量时间依赖型影响;奎巴因可以通过抑制细胞N a ,K -ATPase影响ALR对H epG 2细胞增殖促进作用。因此,N a ,K -ATPase能参与调节ALR促进H epG 2细胞的增殖。     

The influence of trophic level as measured by δ15N on mercury concentrations in freshwater organisms

The relationship between mercury (Hg) concentrations in freshwater biota and trophic position, as defined by stable nitrogen isotope ratios (δ¹⁵N), was examined in 6 lakes in northwestern Ontario. The heavier isotope of nitrogen (¹⁵N) increases an average of 3 parts per thousand (‰) from prey to predator and is used as a measure of an organism's trophic position. Dorsal muscle from lake trout, burbot, walleye, northern pike, white sucker, lake cisco, lake whitefish, and yellow perch was analyzed for Hg and δ¹⁵N using flameless atomic absorption and mass spectrometry respectively. Within each lake, log Hg was significantly related to δ¹⁵N (r ² ranged from 0.47 to 0.91,P<0.01). For four species, yellow perch, northern pike, lake cisco, and lake trout, log Hg was positively related to δ¹⁵N (r ² ranged from 0.37 to 0.47,P≤0.09) across all lakes. We also used δ¹⁵N measurements (assuming a 3‰ shift between an organism and its diet) and the developed within-lake regression equations to calculate a prey Hg for each individual fish. These food Hg values were then used to predict predator Hg using Norstromet al's bioenergetics model. Predicted results were strongly correlated to measured Hg concentrations (r=0.91,P<0.001), indicating that δ¹⁵N has potential to be used in modeling.