MECHANISM OF PHOTOOXYGENATION OF THE CD (II) COMPLEX OF PYROPHEOPHORBIDE A METHYL ESTER

Photooxygenation of (pyropheophorbidato a methyl ester)cadmium (II) was studied using ^18,18O₂ labeling of the molecular oxygen required for cleavage of the macrocycle. After reductive demetallation of the primary oxidation product (4,5-dioxo-4,5-secopyropheophorbidato a methyl ester)cadmium (II), the isotope content of formylbilinone 4a was analyzed by repeated-scan fast atom bombardment mass spectrometry. Comparison of the spectroscopic data of the labeled pigment 4a with the statistical probabilities of¹⁸ O isotope incorporation calculated for four possible reaction mechanisms clearly proves that photooxidative ring cleavage occurred by the one-molecule mechanism, i.e. the terminal oxygen atoms of 4a were derived from one oxygen molecule. Furthermore, a study of the exchange of the¹⁸ O-labeled atoms revealed that no exchange occurs within the pH 4.5–9.5 range. In stronger alkaline or acidic solutions, only the oxygen atom of the formyl group is exchanged. Hydrolysis of the methyl ester group of 4a was achieved, without loss of the¹⁸ O label on the formyl group, at pH 7.2 in the presence of pig liver esterase.     

Fast quality assessment of German chamomile (Matricaria chamomilla L.) by headspace solid-phase microextraction: influence of flower development stage

For an adequate quality evaluation of aromatic plants grown under different conditions, a rapid, simple and sensitive method for the analysis of volatile constituents is indispensable. The main objective of the present study was to compare fast screening of German chamomile (Matricaria chamomilla L.) by means of headspace solid-phase microextraction (HS-SPME) with conventional isolation of the essential oil (steam distillation-solvent extraction (SDSE)) for the differentiation of chamomile essential oil constituents. Flowers were harvested at two distinct development stages: stage I, when ligulate flowers start to develop and tubular flowers are still closed, and stage II, when tubular flowers are partially to completely opened. Dried chamomile flowers at two development stages were extracted by means of both SDSE and HS-SPME, followed by GC-MS analysis. Among 30 compounds detected, (E)-beta-farnesene (49%), artemisia ketone (10%) and germacrene D (9%) were the predominant volatile components in the HS-SPME-extract, while alpha-bisabolol oxide A (42%), chamazulene (21%) and (Z)-spiroether (8%) were the main essential oil constituents among the 13 compounds obtained by SDSE. After statistical analysis of the data, both techniques enabled the same conclusion: (E)-beta-farnesene was the only compound which showed significant differences between the two flower development stages. These results suggest that HS-SPME-GC-MS can be used as a sensitive technique for the rapid screening and quality assessment of M. chamomilla.     

Styrene oxide DNA adducts: quantitative determination using <Superscript>31</Superscript>P monitoring

The reaction of styrene oxide, a potential carcinogen in humans, with DNA constituents has been used to develop an improved method for quantification of DNA adducts. To enable monitoring of DNA adducts caused by xenobiotics at physiological relevant levels, a robust, reliable and powerful method based on monitoring of phosphorus in nucleotides is described. An efficient enzymatic digestion step and a sample-preconcentration procedure are essential, and enable separation of alkylated nucleotides from the large excess of native nucleotides. The adducts are detected by means of the phosphorus signal measured at mass m/z=31 with an inductively-coupled-plasma mass spectrometer. Bis(4-nitrophenyl)phosphate (BNPP) serves as internal standard for quantification of the adducts. The absolute limit of detection, 45 fmol, corresponds to detection of three modified nucleotides among 10⁷ native nucleotides (the calculation is based on use of 50 g calf thymus DNA). An adduct formation ratio at the DNA of 3.6 adducts per 1000 nucleotides was measured, which is 75% lower than for reaction with monomeric 2-deoxy-nucleotides. In addition, a substantial amount of phosphate adducts were detected, but in DNA the rate of phosphate formation was lower than with monomeric nucleotides. Most probably these adducts escaped unnoticed when ³¹P-post-labelling was employed.     

Genotoxicity and Cytotoxicity of 4-Nonylphenol Ethoxylate on Lymphocytes as Assessed by the Comet Assay

Surfactants, which are prevalent at industrial sites and in the environment generally, are potential risk factors in human carcinogenesis. The widespread industrial use of surfactants such as 4-alkylphenol ethoxylates and their prevalence in many cleaning products have provoked studies about surfactant concentrations in water and their toxicity levels. Up to now, these substances have mainly been tested on aquatic organisms. Though tests on human cell lines are rare. The alkaline Comet assay was performed to evaluate the genotoxicity of 4-nonylphenol ethoxylate, a biodegradable product of 4-alkylphenol ethoxylate, in human lymphocytes. Concentrations tested ranged from 0.15 to 150 µg/mL. Test concentrations of 10 to 15 µg/mL caused an increase level of DNA migration in human cells, but without inducing excessive toxicity (viability > 80%). Though induced levels of DNA migration starting at concentrations of 30 µg/mL may have been due to excessive levels of cytotoxicity (viability < 70%). Based on these data, 4-nonylphenol ethoxylate can induce DNA damage in human lymphocytes but at higher concentrations than are normally found in river or drinking water. However, considering the prevalence of surfactants, the measured genotoxicity of these substances is of concern. Further investigations on human target cells are necessary to evaluate the carcinogenic impact of surfactants and reconsider their environmental acceptance.     

Transgenic Expression and Functional Characterization of Human Platelet Derived Growth Factor BB (hPDGF-BB) in Tobacco (Nicotiana tabacum L.)

Recombinant human platelet derived growth factor BB (rhPDGF-BB) is clinically approved for treating diabetic neuropathic ulcers. Plant-based expression systems offer less expensive ways of producing recombinant drugs, which do not require purification for clinical use. From this perspective, rhPDGF-BB is an ideal candidate for expression in plants as it can be applied topically. Here, we report a proof of concept study, in which rhPDGF-BB was expressed in tobacco plants, and its biological activity was tested in vitro. The mature human platelet derived growth factor BB (hPDGF-BB) gene was codon-optimized for tobacco and fused with ER targeting and retention signals, 5′ and 3′ UTRs of arc5-1 gene along with CaMV 35S promoter, and then, transferred by Agrobacterium-mediated transformation. Gene and protein expression of hPDGF-BB were confirmed by PCR and immunoblot studies. Bioactivity of hPDGF-BB expressed protein was determined by in vitro assays such as proliferation and migration in NIH3T3 cells. Our data reveals that total soluble proteins containing hPDGF-BB from transgenic plants showed a 4.5-fold increase in fibroblast proliferation compared to non-transgenic plants. Furthermore, plant-made rhPDGF-BB induced chemotaxis of treated cells and promoted wound healing in vitro. These results clearly demonstrate that functionally active rhPDGF-BB protein can be produced in plants and might have therapeutic benefits.     

Speciation of gadolinium in surface water samples and plants by hydrophilic interaction chromatography hyphenated with inductively coupled plasma mass spectrometry

Hydrophilic interaction chromatography (HILIC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was optimized for speciation analysis of gadolinium-based contrast agents in environmental samples, in particular surface river waters and plants. Surface water samples from the Teltow channel, near Berlin, were investigated over a distance of 5 km downstream from the influx of a wastewater treatment plant. The total concentration of gadolinium increased significantly from 50 to 990 ng?L^?1 due to the influx of the contrast agents. After complete mixing with the river water, the concentration remained constant over a distance of at least 4 km. Two main substances [Dotarem® (Gd-DOTA) and Gadovist® (Gd-BT-DO3A)] have been identified in the river water using standards. A gadolinium-based contrast agent, possibly Gd-DOTA (Dotarem®), was also detected in water plant samples taken from the Teltow channel. Therefore, uptake of contrast agents [Gadovist® (Gd-BTDO3A), Magnevist® (Gd-DTPA), Omniscan® (Gd-DTPA-BMA), Dotarem® (Gd-DOTA), and Multihance® (Gd-BOPTA)] by plants was investigated in a model experiment using Lepidium sativum (cress plants). HILIC–ICP-MS was used for identification of different contrast agents, and a first approach for quantification using aqueous standard solutions was tested. For speciation analysis, all investigated contrast agents could be extracted from the plant tissues with a recovery of about 54 % for Multihance® (Gd-BOPTA) up to 106 % for Gadovist® (Gd-BT-DO3A). These experiments demonstrate that all contrast agents investigated are transported from the roots to the leaves where the highest content was measured.