Genomic damage induced in tobacco plants by chlorobenzoic acids--metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls--Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2-4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose-response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

Fate of deoxynivalenol and deoxynivalenol-3-glucoside during cereal-based thermal food processing: a review study

Deoxynivalenol (DON), the most commonly occurring trichothecene in nature, may affect animal and human health through causing diarrhea, vomiting, gastrointestinal inflammation, and immunomodulation. DON-3-glucoside (DON-3G) as a major plant metabolite of the mycotoxin is another “emerging” food safety issue in recent years. Humans may experience potential health risks by consuming DON-contaminated food products. Thus, it is crucial for human and animal health to study also the degradation of DON and DON-3G during thermal food processing. Baking, boiling, steaming, frying, and extrusion cooking are commonly used during thermal food processing and have promising effects on the reduction of mycotoxins in food. For DON, however, the observed effects of these methods, as reported in numerous studies, are ambiguous and do not present a clear picture with regard to reduction or transformation. This review summarized the influence of thermal processing on the stability of DON and the formation of degradation/conversion products. Besides this, also a release of DON and DON-3G from food matrix as well as the release of DON from DON-3G during processing is discussed. In addition, some conflicting findings as reported from the studies on thermal processing as well as cause-effect relationships of the different thermal procedures are explored. Finally, the potential toxic profiles of DON degradation products are discussed as well when data are available.     

Genetic analysis of metabolic defects in the spontaneously hypertensive rat

Abnormalities in carbohydrate and lipid metabolism are common in patients with essential hypertension and in the spontaneously hypertensive rat (SHR). To identify chromosome regions contributing to this clustering of cardiovascular risk factors in the SHR, we searched for quantitative trait loci (QTL) associated with insulin resistance, glucose intolerance, and dyslipidemia by using the HXB/BXH recombinant inbred (RI) strains. Analysis of variance in RI strains suggested significant effects of genetic factors. A genome screening of the RI strains with more than 700 markers revealed QTL significantly associated with insulin resistance on Chromosomes (Chrs) 3 and 19. The Chr 19 QTL was confirmed by testing a previously derived SHR-19 congenic strain: transfer of a Chr 19 segment delineated by markers D19Rat57 and D19Mit7 from the Brown Norway (BN/Cr) strain onto the genetic background of the SHR/Ola was associated with decreased insulin and glucose concentrations and ameliorated insulin resistance at the tissue level. These findings suggest that closely linked genes on Chr 19, or perhaps even a single gene with pleiotropic effects, influence the clustering of metabolic disturbances in the SHR-BN model.     

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM₁₀ (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination.No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample.Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m³), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice  Prague < Sofia.Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.     

Effect of Selenium Pre-treatment on Antioxidative Enzymes and Lipid Peroxidation in Cd-exposed Suckling Rats

Since there are no data about the protective role of selenium (Se) against cadmium (Cd)-induced oxidative damage in early life, we studied the effect of Se supplementation on antioxidative enzyme activity and lipid peroxidation (through thiobarbituric acid reactive substances; TBARS) in suckling Wistar rats exposed to Cd. Treated animals received either Se alone for 9 days (8 μmol, i.e., 0.6 mg Se as Na₂SeO₃ kg⁻¹ b.w., daily, orally; Se group), Cd alone for 5 days (8 μmol, i.e., 0.9 mg Cd as CdCl₂ kg⁻¹ b.w., daily, orally; Cd group), or pre-treatment with Se for 4 days and then co-treatment with Cd for the following 5 days (Se + Cd group). Our results showed that selenium supplementation, with and without Cd, increased SOD activity in the brain and kidney, but not in the liver and GSH-Px activity across all tissues compared to control rats receiving distilled water. Relative to the Cd group, Se + Cd group had higher kidney and brain SOD and GSH-Px activity (but not the liver), while in the liver caused increased and in the brain decreased TBARS level. These results suggest that Se stimulates antioxidative enzymes in immature kidney and brain of Cd-exposed rats and could protect against oxidative damage.     

Large-scale synthesis of hematoregulatory nonapeptide SK&F 107647 by fragment coupling.

Linear and convergent routes for the large-scale preparation of the hematoregulatory nonapeptide (Glp-Glu-Asp)2-DAS-(Lys)2 (2, SK&F 107647) were investigated. A convergent approach ('3 + 2'-route employing Boc-and benzyl ester protecting groups) was selected for the preparation of multihundred-gram quantities of 2. Key steps were the preparation and the coupling of tripeptide hydrochloride (HCl.H)2-DAS-(Lys(Z)-OBn)2 (6, DAS-2,7-L,L-diaminosuberic acid) and tripeptide Glp-Glu(OBn)-Asp(OBn)-OH (26). Several coupling reagents were investigated in order to reduce the amount of epimerization of this fragment coupling. TDBTU [O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl-1,1,3,3-tetrameth yluronium tetrafluoroborate] was identified as the condensation reagent of choice. Using this synthetic route > 97% pure final product in an overall yield of 35% calculated on di-Boc protected 2,7-L,L-diaminosuberic acid was prepared.     

Expression,glycosylation and secretion of yeast acid phosphatase in hamster BHK cells

The gene PHO5 coding for one of the repressible acid phosphatases of the yeastSaccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human -actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular massM _r=62000, indicating substitution of the polypeptide moiety by 2–3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.Abbreviations APase acid phosphatase - PBS phosphate buffered saline - TBS Tris buffered saline - con A concanavalin A - TCA Tetracarpidium conophorum agglutinin     

Genetic and Biochemical Characterization of Mutants of Bacillus subtilis Defective in Succinate Dehydrogenase

Eleven succinate-accumulating mutants of Bacillus subtilis have been mapped by transformation and transduction crosses and characterized with respect to activities of citric acid cycle enzymes. These mutants could be divided into three genetic groups. Nine of the mutants were found to map between argA and leu in the citF locus. A second group was located between lys-1 and trpC2 and the third group could not be located on the B. subtilis chromosome in extensive transduction crosses. All of the citF mutants lack detectable succinate dehydrogenase activity, whereas both of the other groups show a reduced level of this enzyme. In addition, most of the mutants in the citF locus lack cytochrome a, whereas the level of this cytochrome is normal in the other two groups. A procedure has been devised for the solubilization of the succinate dehydrogenase from the membrane of B. subtilis with the non-ionic detergent Brij 58. Some properties of the soluble and bound forms of succinate dehydrogenase are described.     

Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity

The influence of occupational exposure to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) on DNA damage detected in lymphocytes of exposed people (city policemen) was studied. The cellular susceptibility to the induction of the DNA damage and the repair capacity of exposed donors are presented in comparison with matched controls. Monitoring was performed and blood samples (164 donors) were collected in Prague, Czech Republic, during the winter and summer seasons. The single-cell gel electrophoresis (SCGE) assay with an internal standard was applied to evaluate the DNA damage. A challenging dose of 2Gy of X-rays was used to study cellular capacities. In the results of studies of the DNA damage induced in vivo or as an immediate response to the challenging treatment no significant difference was found between exposed and unexposed subgroups. The percentage of non-repaired X-ray-induced DNA damage (residual damage, RD) overall in both seasons was significantly higher in lymphocytes of policemen exposed to c-PAHs than in matched controls (RD(T-DNA), %DNA in the comet tail: winter 36.4+/-22.1 versus 22.7+/-10.8, p < 0.001; summer 47.7+/-22.9 versus 34.7+/-15.2, p < 0.001). The results suggest that occupational exposure to environmental c-PAHs significantly reduces the cellular capacity to repair the DNA damage induced by a challenging treatment. A significant decrease of repair efficiency in donors occupationally exposed to environmental c-PAHs was also observed when subgroups were stratified according to smoking history. In conclusion, our results suggest that environmental exposure to c-PAHs affects the cellular repair processes and can lead to harmful effects hazardous to human health.     

Cold-active beta-galactosidase from Arthrobacter sp. C2-2 forms compact 660 kDa hexamers: crystal structure at 1.9A resolution

The X-ray structure of cold-active beta-galactosidase (isoenzyme C-2-2-1) from an Antarctic bacterium Arthrobacter sp. C2-2 was solved at 1.9A resolution. The enzyme forms 660 kDa hexamers with active sites opened to the central cavity of the hexamer and connected by eight channels with exterior solvent. To our best knowledge, this is the first cold-active beta-galactosidase with known structure and also the first known beta-galactosidase structure in the form of compact hexamers. The hexamer organization regulates access of substrates and ligands to six active sites and this unique packing, present also in solution, raises questions about its purpose and function. This enzyme belongs to glycosyl hydrolase family 2, similarly to Escherichia coli beta-galactosidase, forming tetramers necessary for its enzymatic function. However, we discovered significant differences between these two enzymes affecting the ability of tetramer/hexamer formation and complementation of the active site. This structure reveals new insights into the cold-adaptation mechanisms of enzymatic pathways of extremophiles.