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.     

土壤微生物产电信号评价芘污染毒性的研究

通过向土壤中加入葡萄糖促进微生物产电过程,研究了芘污染条件下土壤产电变化规律。利用双室微生物燃料电池(Microbial fuel cells,MFCs),实时、连续记录芘污染土壤产电电压。产电110 h后结束MFCs运行,采用循环伏安法检测芘对土壤微生物电化学活性的影响;结合PCR-DGGE及测序技术,分析芘对MFCs阳极表面细菌群落结构的影响。结果显示,MFCs产电电量随芘浓度增加显著降低。循环伏安检测显示芘降低了土壤微生物的电化学活性。DNA序列分析表明,阳极细菌与已报道的产电细菌高度相似,包括Sporolactobacillus、Clostridium、Enterobacter、Bacillus及Ethanoligenens。芘降低了Bacillus丰度。     

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.     

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.     

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.     

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.     

Aggregation of clay in six New Zealand soil types as measured by disaggregation procedures

Measurements were made of the yields and carbon concentrations of clay fractions obtained by a number of disaggregation procedures from samples of a range of New Zealand soils. Disaggregation was carried out in water by: minimal shaking by hand; prolonged mechanical shaking; shaking with sodium resin; ultrasonication; acetylacetone treatment; periodate–borate treatment; peroxidation; and peroxidation followed by citrate-dithionite-bicarbonate treatment. There was virtually no clay yielded by any of the six samples when gently suspended in water. Either a combined hydrogen peroxide and sodium citrate-dithionite-bicarbonate treatment or an exhaustive (10× repeated) hydrogen peroxide treatment produced the highest yield of clay except where ultrasonication of the field moist soil sample brought about strong disaggregation. Considerable physical disruption of primary particles could occur on ultrasonication. Air-drying had a variety of effects. In one notable case with a soil having a highly smectitic clay mineralogy, air-drying led to considerable resistance of the sample against disaggregation into clay. Exhaustive peroxidation was usually highly dispersive but it suppressed clay dispersion in samples of two soils from volcanic ash with allophanic clay mineralogies.There was a wide range between samples of the different soils in the concentrations and amounts of carbon in their clay fractions. A high concentration of carbon was present in clay fractions of all samples after even the most severe chemical treatment, with those of the allophanic soils having the highest concentrations of carbon after these treatments.     

Minirhizotron quantification of soybean root growth as affected by reduced A horizon in soil

Shallow soil A horizon (topsoil) caused by soil erosion and soil movement from cultivation is known to reduce soil and crop productivity. The reduction may be related to limitation of root growth. A field study was conducted to investigate the effects of topsoil thickness on distributions of root density and growth. Soybeans [Glycine max (L.) Merr.] were grown on plots of Mexico silt loam (fine, montmorillonitic, mesic Mollic Endoaqualfs) with topsoil thicknesses of 0, 12.5, 25.0, and 37.5 cm above the Bt horizons. Root density was measured 60 and 90 days after planting using a minirhizotron video‐camera system. Root density was significantly reduced as topsoil thickness decreased from 37.5 to 0 cm. Mean density and net change of the density across profile between 30 and 60 days of growth had a linear function of topsoil thickness. The reduction and lower activity induced by shallow topsoil were attributed to detrimental properties in the Bt horizons. Root distribution pattern and rooting depth were not significantly affected by topsoil thickness. The roots appeared to be accumulated on the upper layers of the Bt horizons. Roots growing in thicker topsoil were more active than roots growing without topsoil. High soil moisture content during the growing season may mitigate the detrimental effects of shallow topsoil, inhibit root penetration, and enhance root activity.     

氮肥优化减施对土壤-莴苣系统氮磷平衡及产量效益的影响

采用田间小区试验, 研究了氮肥减施20%(80%N)与施肥优化[施缓释尿素并减氮20%(80%N+CRU)、氮肥减施20%并添加脲酶抑制剂/硝化抑制剂(80%N+QD)、氮肥减施20%并添加土壤调理剂(80%N+SC)]对土壤-莴苣系统氮磷平衡以及产量效益的影响。结果表明: 3 种优化减氮处理均在一定程度上降低了菜地土壤NO₃^--N(硝态氮)、Olsen-P(有效磷)含量及硝化率水平, 从而降低了氮磷流失进入环境的风险; 同时减氮处理还提高了土壤氮素的利用效率, 增加了蔬菜鲜样产量及菜农实际收入, 效益明显。其中, 80%N+CRU 处理降低土壤NO₃^--N 和Olsen-P 含量最为明显, 但该处理植株可食部分容易累积硝酸盐, 收获时莴苣茎硝酸盐含量显著高于常规施肥处理(CF)和《农产品安全质量无公害蔬菜安全要求》(GB18406.1—2001)(P<0.05); 80%N+QD 处理对各时期土壤Olsen-P 及蔬菜可食部分硝酸盐含量影响较小, 却显著降低了菜地土壤的硝化率水平(P<0.05),从而提高了养分利用效率, 同时与CF 处理相比, 该处理较大幅度增加了莴苣鲜样产量(茎21.7%, 叶7.6%)、总收入(13.0%)、实际收入(14.0%)和产投比(14.2%), 是最佳减氮优化处理。试验地80%N+SC 处理效果不稳定,与常规施肥差异不显著。兼顾经济、食用风险、生态环境等效益, 建议在高肥力菜地土壤中, 氮肥减量与脲酶抑制剂、硝化抑制剂配合施用。     

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.     

Labile pools of copper and zinc in calcareous soils as measured by DTPA and plant extraction

Abstract

Labile pools of Cu and Zn were measured on two calcareous soils from Saudi Arabia, using successive extraction with DTPA, and successive cropping followed by extraction with DTPA after cropping. The examined soils differed in their ability to supply Cu and Zn. The first DTPA extraction removed a major proportion of the labile pool, particularly in the sandy soil. The two soils showed a general decrease in extractable Cu and Zn with progressive extractions, with DTPA extractable Cu declining more rapidly than Zn. DTPA extractable Cu and Zn determined after cropping were highly correlated with DTPA extractable Cu and Zn values obtained after successive extractions. The results gave evidence on both the contribution of element dissolution from insoluble forms with progressive extraction or cropping, and the usefulness of the DTPA extractant for monitoring the availability of Cu and Zn in these calcareous soils.