An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.
Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50?=?478 mg/L vs. fish LC50?=?0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50?=?0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in metabolization.
We investigate evapotranspiration, sap flow and top soil water content variations in a wax myrtle-tree heath (fayal-brezal in Spanish) cloud forest in the Garajonay National Park (La Gomera, Canary Islands) over a 1-year period. We provide transpiration estimates for one of the representative species, the shrubby needle-like Erica arborea L., present in this relict subtropical forest. An ad hoc tree up to the stand scaling method that combines the sap flow and auxiliary reference evapotranspiration data is illustrated, showing to be useful when sap flow in a limited number of trees has been monitored. Individual daily-based scaling curves of the Gompertz type were necessary to explain the observed sap flow variability in E. arborea during the 1-year period investigated (r(2)???0.953 with mode of r(2)?=?0.9999). The mean daily sap flow of an E. arborea individual amounted to 8.37?±?5.65 kg day(-1) tree(-1), with a maximum of 20.48 kg day(-1) tree(-1), yielding an annual total of 3052.89 kg tree(-1). A comparison of the computed daily transpiration with the continuous micrometeorological time series monitored in the studied plot suggested that solar radiation was the main driving force of transpiration in E. arborea (cross correlation index?=?0.94). Fog may also affect tree transpiration via its reduction of radiation and temperature, such that during foggy periods the mean daily water loss estimate of E. arborea was 5.35?±?4.30 kg day(-1) tree(-1), which sharply contrasted with the 2.4-fold average transpiration values obtained for fog-free days, i.e., 12.81?±?4.33 kg day(-1) tree(-1). The annual water balance rendered a 288 mm year(-1) water input to the forest and evidenced the need for accurately quantifying the contribution of fog water dripping from the canopy.
Recent years have seen resurging interest in cancer cell metabolism and the role of secreted cancer metabolites in modulating the tumor stroma. Using a combination of nontargeted and targeted LC and GC-MS methods, the exometabolomes of three leukemia, two melanoma, three renal cell carcinoma, two colorectal adenocarcinoma, four hepatocellular carcinoma, three breast cancer, two bladder carcinoma, and one glioblastoma cell line, as well as five primary cultures of human melanocytes, hepatocytes, monocytes, CD4 and CD8 lymphocytes, that had been all cultivated under identical conditions, were investigated. Unsupervised affinity propagation clustering of the metabolic footprints yielded five distinct clusters that grouped the investigated cell cultures mainly according to the tissue of origin. A common expected feature of all neoplastic cells was high lactate production. Extracellular arginine and nicotinamide were major discriminants between normal and neoplastic hepatocytes. Further, significant differences in the assimilation of di- and tripeptides were observed. This finding appears to underscore the importance of peptides for meeting the increased bioenergetic and biosynthetic demands of many cancers.
The ecophysiologic role of fog in the evergreen heath-laurel laurisilva cloud forests of the Canary Islands has not been unequivocally demonstrated, although it is generally assumed that fog water is important for the survival and the distribution of this relict paleoecosystem of the North Atlantic Macaronesian archipelagos. To determine the role of fog in this ecosystem, we combined direct transpiration measurements of heath-laurel tree species, obtained with Graniers heat dissipation probes, with micrometeorological and artificial fog collection measurements carried out in a 43.7-ha watershed located in the Garajonay National Park (La Gomera, Canary Islands, Spain) over a 10-month period. Median ambient temperature spanned from 7 to 15 degrees C under foggy conditions whereas higher values, ranging from 9 to 21 degrees C, were registered during fog-free periods. Additionally, during the periods when fog water was collected, global solar radiation values were linearly related (r2=0.831) to those under fog-free conditions, such that there was a 75+/-1% reduction in median radiation in response to fog. Fog events greatly reduced median diurnal tree transpiration, with rates about 30 times lower than that during fog-free conditions and approximating the nighttime rates in both species studied (the needle-like leaf Erica arborea L. and the broadleaf Myrica faya Ait.). This large decrease in transpiration in response to fog was independent of the time of the day, tree size and species and micrometeorological status, both when expressed on a median basis and in cumulative terms for the entire 10-month measuring period. We conclude that, in contrast to the turbulent deposition of fog water droplets on the heath-laurel species, which may be regarded as a localized hydrological phenomenon that is important for high-altitude wind-exposed E. arborea trees, the cooler, wetter and shaded microenvironment provided by the cloud immersion belt represents a large-scale effect that is crucial for reducing the transpirational water loss of trees that have profligate water use, such as those of the laurisilva.
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