Comparison of the molecular effects of the mycotoxins beta-zearalenol and deoxynivalenol in porcine endometrial cells--a review

The mycotoxins beta-zearalenol (beta-ZOL) and deoxynivalenol (DON) produce toxic effects that result in diseases in humans and animals. The molecular mechanisms that control the mycotoxin-mediated effects are far from being completely understood. Various results show that these mycotoxins could inhibit cell proliferation. In the present short communication, the influence of beta-ZOL and DON on the abundance and phosphorylation state of kinases that are included in regulation of the initiation of mRNA translation (which is correlated with cell proliferation) was compared in porcine endometrial cells (PEC). Our results indicate that these mycotoxins modulate the expression and phosphorylation of these factors in a different manner. Whereas beta-ZOL mainly had an impact on the biological activity of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (Akt), eukaryotic initiation factor 4E (eIF4E) and its repressor 4E binding protein 1 (4E-BP1), DON reduced the abundance of p38 MAPk, Akt and specific 4E-BP1 bands. In summary, these results indicate that beta-ZOL influences molecular events that are included in the initiation of mRNA translation in the porcine endometrium but DON does not alter such processes clearly.     

Contrasting response of two forest soils to nitrogen input: rapidly altered NO and N2O emissions and nirK abundance

Denitrification represents one of the main microbial processes producing the primary and secondary greenhouse gases nitrous oxide (N₂O) and nitric oxide (NO) in soils. It is well established that abiotic factors like the soil water content and the availability of nitrogen (N) are key parameters determining the activity of denitrifiers in soils. However, soils differing regarding their characteristics such as the content of C_org, the soil texture or the pH value may respond in specific manners to equivalent changes in soil moisture and N input. Thus, short-term incubation experiments were performed to test and compare the capacity of two contrasting Austrian forest soils to respond to mineral N application at increased soil water contents. Soils from the pristine Rothwald forest (rich in C_org) and the more acidic Schottenwald forest (poor in C_org) were amended with either NH ₄ ⁺ -N or NO ₃ ^? -N and were incubated at 40% and 70% water-filled pore space for 4 days. Changes in mineral N pools, nitrite reductase activity and NO and N₂O emission rates were measured, and the abundance and structural community composition of the functional group involved in nitrite reduction were analysed via quantitative real-time polymerase chain reaction and terminal restriction fragment length polymorphism analysis of the nirK gene. Rapid and distinct activity responses to increased soil moisture and altered mineral nitrogen availability were observed in two contrasting forest soils. In both soils, nitrogen oxide emission rates were stimulated by N inputs and, depending on the soil moisture status, either NO or N₂O emission was prevailing. However, different N cycling processes appeared to predominate in either soil under equivalent treatment. Nitrogen oxide emissions peaked following NO ₃ ^? application in Schottenwald soils but were the highest after NH ₄ ⁺ application in Rothwald soils. Denitrifying (nirK) communities differed significantly in Rothwald and Schottenwald soils; however, changes in the community structure were marginal during the short-term incubation. Abundances of nirK genes remained unaffected by N application in either soil. The soil water content affected nirK gene abundances only in Rothwald soil, indicating a distinct reaction of nitrite reducing communities in the two soils.     

The contribution of lateral aquatic habitats to insect diversity along river corridors in the Alps

River floodplains are among the most threatened ecosystems globally. Understanding the mechanisms that create and maintain biodiversity and ecosystem functioning of floodplains, is therefore a prerequisite for developing scientifically-sound management and conservation strategies. We quantified the spatial distribution of lateral aquatic habitats (i.e. tributaries, ponds, backwaters) and the associated insect assemblages (Ephemeroptera, Plecoptera, Trichoptera) along three Alpine river corridors (Tagliamento, Thur, and Rhône). Our objective was to assess the relative contribution of lateral habitats to river corridor diversity, and to identify the scale that contributed the most to regional (Alpine scale) species diversity. The number of lateral habitats decreased by 72 % from the near-natural Tagliamento (101) to the Thur river (42) and the regulated Rhône river (28). More than 50 % of the total species richness along each river was restricted to lateral habitats, which also exhibited higher taxon turnover rates (Whittaker’s beta diversity) than the associated main channel. Hierarchical diversity partitioning revealed that beta diversity among corridors was higher than expected, and accounted for 48 % of regional richness, partly reflecting biogeographical differences among catchments. However, diversity partitioning, excluding catchment-specific effects, showed that beta diversity among habitat types contributed significantly to regional richness (79 %). The present study is among the first to quantify the distribution and biodiversity of floodplain habitats along entire river continua. Our results demonstrated that biodiversity would be best preserved by protecting multiple catchments, and that lateral floodplain habitats contribute disproportionally to species richness at the river corridor and regional scale.     

Combined effects of the antibiotic sulfadiazine and liquid manure on the soil microbial‐community structure and functions

Sulfonamides are the second most used antibiotic class in veterinary medicine and applied to livestock to treat bacterial infections. Subsequently, they are spread onto agricultural soils together with the contaminated manure used as fertilizer. Both manure and antibiotics affect the soil microbial community. However, the influence of different liquid manure loads on effects of antibiotics to soil microorganisms is not well understood. Therefore, we performed a microcosm experiment for up to 32 d to clarify whether the function and structure of the soil microbial community is differently affected by interactions of manure and the antibiotic sulfadiazine (SDZ). To this end selected concentrations of pig liquid manure (0, 20, 40, 80 g kg^–1) and SDZ (0, 10, 100 mg kg^–1) were combined. We hypothesized that incremental manure amendment might reduce the effect of SDZ in soils, due to an increasing sorption capacity of SDZ to organic compounds. Clear dose‐dependent effects of SDZ on microbial biomass and PLFA pattern were determined, and SDZ effects interacted with the liquid manure application rate. Soil microbial biomass increased with incremental liquid manure addition, whereas this effect was absent in the presence of additional SDZ. However, activities of enzymes such as urease and protease were only slightly affected and basal respiration was not affected by SDZ application, while differences mostly depended on the concentration of liquid manure. These results illustrated that the microbial biomass and structural composition react more sensitive to SDZ contamination than functional processes. Furthermore, effects disproportionally increased with incremental liquid manure addition, although extractable amounts of SDZ declined with increasing liquid manure application.     

The impact of extraction methodologies on the toxicity of sediments in the zebrafish (Danio rerio) embryo test

Purpose

Traditionally, methods for sediment extractions are characterised using chemical analyses. However, in order to evaluate sediment extracts with regard to biological effects and, thus, bioaccessibility, extraction methods have to be compared to effect data obtained from experiments with in situ exposure scenarios, i.e., sediment contact tests. This study compares four extraction methods and sediment contact test data from a previous project with respect to predictive power in the fish embryo test with zebrafish (Danio rerio).

Materials and methods

A natural and an artificial sediment spiked with a mixture of six organic pollutants (2,4-dinitrophenol, diuron, fluoranthene, nonylphenol, parathion and pentachlorophenol) were extracted using (a) membrane dialysis extraction (MDE), (b) a Soxhlet procedure, (c) hydroxypropyl-??-cyclodextrin (HPCD) or (d) Tenax^®-TA. Whereas the former two are regarded being exhaustive with respect to non-covalently bound contaminants, the latter two are considered to predict bioaccessibility. Resulting extracts were tested in the fish embryo assay with D. rerio for embryotoxic and teratogenic potential.

Results and discussion

Mortalities caused by organic extracts from Soxhlet extraction and MDE were high. However, HPCD extracts turned out to be at least as effective as extracts obtained with these two methods. One possible reason might be short ageing time of the spiked sediments. Only Tenax^®-TA extracts gave results comparable to the sediment contact assay for natural sediment, but revealed low reproducibility. Significant differences between natural and artificial sediment were found for extracts obtained with techniques using native (i.e., non-freeze-dried) sediments, i.e., HPCD and Tenax^®-TA. In contrast, MDE and Soxhlet extracts used freeze-dried sediment and did not differentiate between natural and artificial sediment. Therefore, freeze-drying has likely altered and equalised sediment properties that influence accessibility, such as composition of bacterial communities and organic matter quality.

Conclusions

Four extraction methods were successfully characterised with respect to their stringency and predictiveness for bioaccessibility. MDE was confirmed as an alternative to Soxhlet extraction. High mortalities induced by HPCD extracts underline the need to include ageing into consideration when assessing sediments. Although Tenax^®-TA may basically be used to predict bioaccessibility in the fish embryo test, the high variability observed warrants further investigation of the relation between effect and extractability. Apparently, freeze-drying can severely affect sediment properties, potentially eliminating individual properties of natural sediments.     

Contribution of anecic earthworms to biopore formation during cultivation of perennial ley crops

Large sized biopores (diameter >2 mm) in the subsoil can be created by tap roots, which leave voids after their decay, or by the burrowing activity of anecic earthworms which may benefit from the temporary lack in tillage in perennial cropping systems. However, the interactions between root growth and earthworm activity in the process of biopore formation during perennial ley cropping are not well understood. The aim of this field study was to quantify the development of the abundance of the anecic earthworm Lumbricus terrestris and the biopore density during the cultivation of lucerne (Medicago sativa L.), chicory (Cichorium intybus L.) and tall fescue (Festuca arundinacea Schreb.) grown for either one, two or three years. An increased abundance of L. terrestris was already recorded after two years of continuous ley when compared with one year cultivation. The ley crop species had only minor influence on the abundance of L. terrestris. Biopore densities of both diameter classes under study (2–5 mm and >5 mm) were not significantly affected by the duration of ley cropping. In contrast, biopore densities were influenced by ley crop species. More biopores of diameter class 2–5 mm were recorded after chicory than after fescue. Lucerne cropping resulted in intermediate biopore density. Additionally, in an incubation experiment under field conditions, we quantified whether L. terrestris preferentially created new biopores or colonized abandoned, previously existing ones. After three weeks of incubation, one third of the adult individuals incubated in the experimental area created new biopores at 0.4 m soil depth. A similar percentage of individuals colonized previously existing biopores, partially widening the lumen of smaller sized biopores. The remaining individuals remained in the topsoil. Sub-adult individuals rarely formed new pores. Half of the introduced sub-adults remained in the topsoil. We conclude that in crop rotations new biopores can be generated during perennial ley cropping by taproot systems of ley crops, but that a two to three- year period without tillage is not sufficient for populations of anecic earthworms to make a marked contribution to biopore density in the subsoil. The relevance of anecic earthworms for altering physical and chemical properties of biopores during ley cropping still needs further investigation.     

Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella fijiensis field isolates

Field isolates of Mycosphaerella fijiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc₁ enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin‐resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross‐resistance was observed among all Qo inhibitors, including trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa₃ inhibitors, antimycin A and potassium cyanide, respectively, were not cross‐resistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell‐free NADH‐consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub‐mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by trifloxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M fijiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the field isolates of M fijiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non‐mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance. © 2000 Society of Chemical Industry