Consumer Risk Assessment Associated with Intake of Pesticide Residues in Food of Plant Origin from the Retail Market in Poland

The aim of this study was to characterize short- and long-term risk for consumers associated with dietary intake of pesticide residues in fruits, vegetables, and other foodstuffs available on the Polish market based on 2010–2013 official surveillance results. Among 779 samples collected from 2010 to 2013 no pesticide residue was found in 39.7% samples while 58.5% contained residues at or below the EU Maximum Residue Levels (MRLs). Non-compliances (residues above the respective MRLs) were found in 14 samples (1.8%). Most of the estimated daily intakes were well below 1% of respective acceptable daily intake (ADI) values. The highest intake for children and adults was about 7% and 1.5% of ADI, respectively. For non-compliant results acute risk was characterized. Predicted short-term intakes for children and adults ranged from 0.7% to 425%, and from 0.2% to 100% of respective acute reference dose, respectively. Results of chronic risk characterization show that consumers in Poland are adequately protected; however, incidental cases where residue levels may potentially pose a threat to consumers’ health due to acute exposure cannot be excluded.     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

Vacuolating,lethal, collagenase and clostripain activities of six reference ATCC <Emphasis Type="Italic">Clostridium histolyticum</Emphasis> strains

We have previously demonstrated that C. histolyticum reference strain ATCC 19401 produces not only lethal factor but also hitherto unrecognized vacuolating toxin. The aim of this study was to compare vacuolating and lethal activities of six reference C. histolyticum strains (ATCC 6282, 8034, 17859, 17860, 19401 and 25770) and to determine whether production of vacuolating toxin is strain-dependent and how the amounts of both toxins produced by the same strain are related to each other and also to protease, collagenase and clostripain activities. All strains produced vacuolating and lethal toxins as well as collagenase, clostripain and proteases, but with different yield. Strain ATCC 19401 demonstrated considerable vacuolating and lethal activities and low activity of collagenase, clostripain and proteases. In other strains such relationship was not evident. Positive correlations were observed in collagenase and clostripain activities of all studied C. histolyticum strains (r = 0.71). Positive correlations were detected also in vacuolating activities of studied strains and clostripain (r = 0.62) and collagenase (r = 0.87) production, and this effect was statistically significant (P < 0.05). Negative non-significant correlations were detected: (a) in lethal activities of studied strains and clostripain, or collagenase activities, (b) in vacuolating activities and protease production.     

Arene- and quinoline-sulfonamides as novel 5-HT7 receptor ligands

Novel arene- and quinolinesulfonamides were synthesized using different solutions and a solid-support methodology, and were evaluated for their affinity for 5-HT(1A), 5-HT(2A), 5-HT(6), and 5-HT(7) receptors. Compound 54 (N-Ethyl-N-[4-(1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinolin-2-yl)butyl]-8-quinolinesulfonamide) was identified as potent 5-HT(7) antagonist (K(i)=13 nM, K(B)=140 nM) with good selectivity over 5-HT(1A), 5-HT(2A), 5-HT(6) receptors. In the FST in mice, it reduced immobility in a manner similar to the selective 5-HT(7) antagonist SB-269970.     

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.     

A new way of solid-phase microextraction fibers preparation for selected antibiotic drug determination by HPLC-MS

The polypyrrole (PPy) and polythiophene (PTh) solid phase microextraction (SPME) coatings were obtained with the use of the electropolymerisation and linear sweep voltammetry. Such fibers were modified by an ozone treatment in a gaseous phase in the concentration of 2.1 ± 0.2 × 10(-5) mol dm(-3). Both kinds of fibers were applied in the microextraction of linezolid from standard solutions to compare the extraction efficiencies displayed by these sorption phases. In these investigations a better adsorption capacity was obtained for polypyrrole fibers and hence only these kinds of fibers were utilized in the measurements from human plasma. In all measurements the concentrations of the drugs were in the range from 1 to 20 μg ml(-1) (standard solutions) and 1 to 15 μg ml(-1) (human plasma). Before the measurements, an optimization of the desorption solution experiments was performed. The correlation coefficients (R) obtained in the standard solution and human plasma were in the range from 0.8399 to 0.9970. The relative standard deviations (RSDs) were in the range of 0.1-7.6%.     

Applications of the ETS-NOCV method in descriptions of chemical reactions

The present study characterizes changes in the electronic structure of reactants during chemical reactions based on the combined charge and energy decomposition scheme, ETS-NOCV (extended transition state–natural orbitals for chemical valence). Decomposition of the activation barrier, ΔE ^#, into stabilizing (orbital interaction, ΔE _orb, and electrostatic, ΔE _elstat) and destabilizing (Pauli repulsion, ΔE _Pauli, and geometry distortion energy, ΔE _dist) factors is discussed in detail for the following reactions: (I) hydrogen cyanide to hydrogen isocyanide, HCN → CNH isomerization; (II) Diels-Alder cycloaddition of ethene to 1,3-butadiene; and two catalytic processes, i.e., (III) insertion of ethylene into the metal-alkyl bond using half-titanocene with phenyl-phenoxy ligand catalyst; and (IV) B–H bond activation catalyzed by an Ir-containing catalyst. Various reference states for fragments were applied in ETS-NOCV analysis. We found that NOCV-based deformation densities (Δρ _i) and the corresponding energies ΔE _orb(i) obtained from the ETS-NOCV scheme provide a very useful picture, both qualitatively and quantitatively, of electronic density reorganization along the considered reaction pathways. Decomposition of the barrier ΔE^# into stabilizing and destabilizing contributions allowed us to conclude that the main factor responsible for the existence of positive values of ΔE ^# for all processes (I, II, III and IV) is Pauli interaction, which is the origin of steric repulsion. In addition, in the case of reactions II, III and IV, a significant degree of structural deformation of the reactants, as measured by the geometry distortion energy, plays an important role. Depending on the reaction type, stabilization of the transition state (relatively to the reactants) originating either from the orbital interaction term or from electrostatic attraction can be of vital importance. Finally, use of the ETS-NOCV method to describe catalytic reactions allows extraction of information on the role of catalysts in determination of ΔE ^#.     

ATP and its N<Superscript>6</Superscript>-substituted analogues: parameterization,molecular dynamics simulation and conformational analysis

In this work we used a combination of classical molecular dynamics and simulated annealing techniques to shed more light on the conformational flexibility of 12 adenosine triphosphate (ATP) analogues in a water environment. We present simulations in AMBER force field for ATP and 12 published analogues [Shah et al. (1997) Proc Natl Acad Sci USA 94: 3565–3570]. The calculations were carried out using the generalized Born (GB) solvation model in the presence of the cation Mg²⁺. The ion was placed at a close distance (2 Å) from the charged oxygen atoms of the beta and gamma phosphate groups of the ?3 negatively charged ATP analogue molecules. Analysis of the results revealed the distribution of inter-proton distances H8–H1′ and H8–H2′ versus the torsion angle ψ (C4–N9-C1′–O4′) for all conformations of ATP analogues. There are two gaps in the distribution of torsion angle ψ values: the first is between ?30 and 30 degrees and is described by cis-conformation; and the second is between 90 and 175 degrees, which mostly covers a region of anti conformation. Our results compare favorably with results obtained in experimental assays [Jiang and Mao (2002) Polyhedron 21:435–438].

Open image in new window

Figure Dihedral O4′–C1′–N9–C4 angle dependence on inter-proton distances H8–H1′ (crosses) and H8–H2′ (dots) measured for ATP

     

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.