The use of comet assay data with a simple reaction mechanism to evaluate the relative effectiveness of free radical scavenging by quercetin, epigallocatechin gallate and N-acetylcysteine in UV-irradiated MRC5 lung fibroblasts

Comet assay data (tail DNA %) have been gathered for the concentration dependent role of three antioxidants (AOs); quercetin (Q), epigallocatechin gallate (EGCG) and N-acetylcysteine (NAC) in reducing UV-induced damage to DNA in normal fetal lung fibroblasts (MRC5). All three compounds demonstrate a concentration dependent reduction maximum with a pro-oxidant effect at higher (though not cytotoxic) concentrations. Manipulation of a simple 4-step reaction mechanism for free radical (FR) scavenging by AOs produced rate constant ratios which allowed the relative effectiveness (Q > EGCG > NAC) of the AOs to be evaluated.     

Interpretation of CALUX results in view of the EU maximal TEQ level in milk

Analyses of dioxins in food have become increasingly important since the European Commission has enforced maximal toxic equivalent concentration (TEQ) levels in various food and feed products. Screening methodologies are usually used to exempt those samples that are below the maximum permitted limit and that can, therefore, be released to the market. In addition, one needs to select those samples that require confirmation of their dioxin TEQ level. When bioassays are used as screening tools, the interpretation of the obtained results should consider the higher variability and uncertainty associated with them. This paper explores the use of CALUX data as quantitative screening results. The validation of the method for the polychlorinated dibenzo-p-dioxins (PCDD)/F TEQ determination in milk samples is described with emphasis on the decision limit (CC) and the precision of the method. The decision limit amounts to 4.53 pg TEQ/g fat. Repeatability and within-lab reproducibility coefficients of variation are below 30%. The newly introduced parameter CC^* of 1.47 pg TEQ/g fat delimits with CC a range of suspicious results. These data are not significantly different from the maximum limit of 3 pg TEQ/g fat and should be confirmed by a confirmatory analytical method such as HRGC–HRMS.     

The bimolecular hydrogen-deuterium exchange behavior of protonated alkyl dipeptides in the gas phase

As part of an ongoing characterization of the intrinsic chemical properties of peptides, thermal hydrogen-deuterium exchange has been studied for a series of fast-atom-bombardment-generated protonated alkyldipeptides and related model compounds in the reaction with D₂O, CH₃OD, and ND₃ in a Fourier transform ion cyclotron resonance mass spectrometer. Despite the very large basicity difference between the dipeptides and the D₂O and CH₃OD exchange reagents, efficient exchange of all active hydrogen atoms occurs. From the kinetic data it appears that exchange of the amino, amide, and hydroxyl hydrogens proceeds with different efficiencies, which implies that the proton in thermal protonated dipeptides is immobile. The selectivity of the exchange at the different basic sites is governed by the nature of both the dipeptide and the exchange reagent. The results indicate that reversible proton transfer in the reaction complexes, which effectuates the deuterium incorporation, is assisted by formation of multiple hydrogen bonds between the reagents. Exchange is considered to proceed via the intermediacy of different competing intermediate complexes, each of which specifically leads to deuterium incorporation at different basic sites. The relative stabilization of the competing intermediate complexes can be related to the relative efficiencies of deuterium incorporation at different basic sites in the dipeptide. For all protonated dipeptides studied, the exchange in the reaction with ND₃ proceeds with unit efficiency, whereas all active hydrogen atoms are exchanged equally efficiently. Evidently specific multiple hydrogen bond formations are far less important in the reversible proton transfers with the relatively basic ammonia, which allows effective randomization of all active hydrogen atoms in the reaction complexes.     

Novel coumarin derivatives containing 1,2,4-triazole, 4,5-dicyanoimidazole and purine moieties: synthesis and evaluation of their cytostatic activity

We report here on the synthesis and in vitro anti-tumor effects of a series of novel 1,2,4-triazole (compounds 3-6), 4,5-dicyanoimidazole (compound 7), and purine (compounds 8-13) coumarin derivatives and their acyclic nucleoside analogues 14-18. Structures of novel compounds 3-18 were deduced from their ¹H- and ¹³C-NMR and corresponding mass spectra. Results of anti-proliferative assays performed on a panel of selected human tumor cell lines revealed that compound 6 had moderate cytostatic activity against the HeLa cell line (IC₅₀ = 35 μM), whereas compound 10 showed moderate activity against the HeLa (IC₅₀ = 33 μM), HepG2 (IC₅₀ = 25 μM) and SW620 (IC₅₀ = 35 μM) cell lines. These compounds showed no cytotoxic effects on normal (diploid) human fibroblasts.     

Bond dissociation energies and radical stabilization energies associated with model peptide-backbone radicals

Bond dissociation energies (BDEs) and radical stabilization energies (RSEs) have been calculated for a series of models that represent a glycine-containing peptide-backbone. High-level methods that have been used include W1, CBS-QB3, U-CBS-QB3, and G3X(MP2)-RAD. Simpler methods used include MP2, B3-LYP, BMK, and MPWB1K in association with the 6-311+G(3df,2p) basis set. We find that the high-level methods produce BDEs and RSEs that are in good agreement with one another. Of the simpler methods, RBMK and RMPWB1K achieve good accuracy for BDEs and RSEs for all the species that were examined. For monosubstituted carbon-centered radicals, we find that the stabilizing effect (as measured by RSEs) of carbonyl substituents (CX=O) ranges from 24.7 to 36.9 kJ mol(-1), with the largest stabilization occurring for the CH=O group. Amino groups (NHY) also stabilize a monosubstituted alpha-carbon radical, with the calculated RSEs ranging from 44.5 to 49.5 kJ mol(-1), the largest stabilization occurring for the NH2 group. In combination, NHY and CX=O substituents on a disubstituted carbon-centered radical produce a large stabilizing effect ranging from 82.0 to 125.8 kJ mol(-1). This translates to a captodative (synergistic) stabilization of 12.8 to 39.4 kJ mol(-1). For monosubstituted nitrogen-centered radicals, we find that the stabilizing effect of methyl and related (CH2Z) substituents ranges from 25.9 to 31.7 kJ mol(-1), the largest stabilization occurring for the CH3 group. Carbonyl substituents (CX=O) destabilize a nitrogen-centered radical relative to the corresponding closed-shell molecule, with the calculated RSEs ranging from -30.8 to -22.3 kJ mol(-1), the largest destabilization occurring for the CH=O group. In combination, CH2Z and CX=O substituents at a nitrogen radical center produce a destabilizing effect ranging from -19.0 to -0.2 kJ mol(-1). This translates to an additional destabilization associated with disubstitution of -18.6 to -7.8 kJ mol(-1).     

Conductance study of association in aqueous CaCl2, Ca(CH3COO)2, and Ca(CH3COO)2.nCH3COOH from 348 to 523 K at 10 MPa

The conductances of aqueous CaCl2, Ca(CH3COO)2, and Ca(CH3COO)2.nCH3COOH were measured from 348 to 523 K at pressures near 10 MPa using a flow conductometric technique. The first association constant for calcium chloride and the first and second association constants for calcium acetate in water, were derived from a least-squares fit of the measurements to a conductance model in which activity coefficients were calculated using the mean spherical approximation (MSA) and the conductance was modeled by the Turq, Blum, Bernard, and Kunz (TBBK) equation, plus a mixture model.     

Poly(ethylene glycol)--oligolactates with monodisperse hydrophobic blocks: preparation, characterization, and behavior in water

Methoxypoly(ethylene glycol)-b-oligo-L-lactate (mPEG-b-OLA) diblock oligomers with monodisperse OLA blocks were obtained by fractionation of polydisperse block oligomers using preparative HPLC. The fractionated oligomers were composed of an mPEG block with a molecular weight of 350, 550, or 750 and an OLA block with a degree of polymerization of 4, 6, 8, or 10. The diblock oligomers with a low PEG content were fully amorphous, with glass transition temperatures ranging from -60 to -20 degrees C, indicating that the blocks were miscible. Upon heating aqueous dispersions of the block oligomers, cloud points, depending on the PEG/OLA ratio of the block oligomer, were observed at temperatures above 40 degrees C. The monodispersity of the hydrophobic block enabled the amphiphilic molecules to form nanoparticles in water with a hydrodynamic radius of 130-300 nm, at concentrations above the critical aggregation concentration (0.4-1 mg/mL), whereas polydisperse mPEG-b-OLAs gave formation of large aggregates. Static light scattering measurements showed that the nanoparticles have a low density (0.6-25 mg/mL), indicating that the particles are highly hydrated. In agreement herewith, the (1)H NMR spectra of nanoparticles in D2O closely resembled spectra in a good solvent for both blocks (CDCl3). It is therefore suggested that the nanoparticles contain a hydrated core of mPEG-b-OLA block oligomers, stabilized by a thin outer PEG layer. The particles were stable for two weeks, except for the mPEG350 series and mPEG750-b-OLA4, indicating that both the PEG block size and the PEG weight fraction of the oligomers determine their stability. The evident self-emulsifying properties of mPEG-b-oligo-l-lactates with monodisperse hydrophobic blocks as demonstrated in this study, together with their expected biocompatibility and biodegradability, make these systems well suitable for pharmaceutical applications.     

Suicide inactivation of dioldehydratase by glycolaldehyde and chloroacetaldehyde: an examination of the reaction mechanism

High-level ab initio calculations have been used to study the mechanism for the inactivation of diol dehydratase (DDH) by glycolaldehyde or 2-chloroacetaldehyde. As in the case of the catalytic substrates of DDH, e.g., ethane-1,2-diol, the 5'-deoxyadenosyl radical (Ado*) is able to abstract a hydrogen atom from both substrate analogues in the initial step on the reaction pathway, as evidenced by comparable energy barriers. However, in subsequent step(s), each substrate analogue produces the highly stable glycolaldehyde radical. The barrier for hydrogen atom reabstraction by the glycolaldehyde radical is calculated to be too high ( approximately 110 kJ mol-1) to allow Ado* to be regenerated and recombine with the cob(II)alamin radical, the latter therefore remaining tightly bound to DDH. As a consequence, the catalytic pathway is disrupted, and DDH becomes an impotent enzyme. Interconversion of equivalent structures of the glycolaldehyde radical via the symmetrical cis-ethanesemidione radical is calculated to require 38 kJ mol-1. EPR indications of a symmetrical cis-ethanesemidione structure are likely to be the result of formation of an equilibrium mixture of glycolaldehyde radical structures, this equilibration being facilitated by partial deprotonation of the glycolaldehyde radical by the carboxylate of an amino acid residue within the active site of DDH.