Zbigniew Wieczorek

Natural Compounds in the Human Diet and their Ability to Bind Mutagens Prevents DNA–Mutagen Intercalation

Human diet may contain many mutagenic or carcinogenic aromatic compounds as well as some beneficial physiologically active dietary components, especially plant food phytochemicals, which act as mutagenesis or carcinogenesis inhibitors. This study compared the binding properties of natural compounds in the human diet (caffeine, theophylline, theobromine, and resveratrol) with a water-soluble derivative of chlorophyll to bind to acridine orange, a known mutagen. An analysis was conducted to determine which substances were effective binding agents and may thus be useful in prevention of chemical-induced mutagenesis and carcinogenesis. Data indicated that in order to bind 50% of the mutagen in a complex, less than twice the concentration of chlorophyllin was needed, the resveratrol concentration was 20-fold higher, while a 1000-fold or even 10,000-fold excess of xanthines were required to bind acridine orange.

Interactions of chlorophyllin with acridine orange, quinacrine mustard and doxorubicin analyzed by light absorption and fluorescence spectroscopy

The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.

Attenuation of acridine mutagen ICR-191 — DNA interactions and DNA damage by the mutagen interceptor chlorophyllin

We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

Fluorescence Studies on Association of Human Translation Initiation Factor eIF4E with mRNA cap-Analogues

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5′-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence ΔFmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E -7- methyl-GDP complexes in crystal and solution.

The Cu2+-Promoted Cleavage of mRNA 5′-cap Analogs: A Kinetic Study with P1-(7-Methylguanosin-5′-yl) P3-(Nucleosid-5′-yl) Triphospates and P1-(7-Methylguanosin-5′-yl) P4-(Guanosin-5′-yl) Tetraphosphate

Abstract A kinetic study on the cleavage of a number of mRNA 5′-cap analogs, m7GpppN and m7GppppG, with Cu2+ aquo ion has been performed. Time-dependent product distributions at various pH and metal ion concentrations have been determined by capillary zone electrophoresis, and these data have been used to calculate the rate constants for various parallel reactions of the breakdown of the cap analogs.

Uptake and phytotoxicity of anthracene and benzo[k]fluoranthene applied to the leaves of celery plants (Apium graveolens var. secalinum L.)

The above-ground parts of celery plants were exposed to two polycyclic aromatic hydrocarbons (PAHs): 3-ring anthracene (ANT) and 5-ring benzo[k]fluoranthene (BkF), and the combination of ANT and BkF. After 43 days of exposure (overall dose of 1325µg/plant), celery plants retained only 1.4% of the total dose of ANT and 17.5% of the total dose of BkF. After exposure to a combination of ANT and BkF (1325µg of each compound per plant), the average ANT concentrations were more than twofold higher in/on leaf blades, whereas BkF levels were insignificantly higher. Under natural photoperiod conditions equivalent to a normal day, the combined application of ANT and BkF to the above-ground parts of celery plants slowed down physicochemical transformations of ANT. A similar effect was observed when PAHs were applied to glass surfaces. The combination of both PAHs probably led to stacking interactions, which decreased volatilization, in particular of ANT. Phytotoxicity of ANT and BkF could not be unambiguously established based on the results of this study. In all analyzed treatments, the chlorophyll content of leaf blades remained unchanged. Foliar application of ANT reduced ascorbic acid levels in all analyzed plant parts and increased the total acidity of celery leaves. In all experimental treatments, the total phenolic content of leaves increased up to 15%. Interestingly, ANT and BkF did not produce cumulative effects when applied in combination (when total PAH concentrations per plant were twofold higher).

Synthesis and evaluation of fluorescent cap analogues for mRNA labelling

We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-β methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins.

Synthesis and Properties of mRNA 5′-Cap Analogues with 7-Methylguanine Replaced by Benzimidazole or 3-Methylbenzimidazole

Abstract Several new analogues of the mRNA 5′-cap structure, m7G(5′)Pn(5′)N, with n = 2–4, have been synthesized in which the m7G component is replaced by 1-(β-D-ribofuranosyl)benzimidazole (RBz) or 3-methyl-RBz. The latter, like m7G, has a positively charged imidazole ring and is likewise fluorescent. All compounds have been characterized by various physico-chemical and enzymatic criteria, and by 1H and 31P NMR spectroscopy.

Electrochemical and spectroscopic study of redox transformation of the 7-methylguanine analog of coenzyme NAD+

Abstract Two NAD + analogs in which the adenine moiety is substituted by 7-methyl-guanine and guanine respectively (Nm 7 GD + and NGD + ) were examined electrochemically, enzymatically and by UV and fluorescence spectroscopy. The enzymatic activity of Nm 7 GD + and NGD + with yeast alcohol dehydrogenase was studied. The kinetic parameters K m and V max for the enzymatic reduction of the two compounds were determined. The electrochemical reduction of Nm 7 GD + and NGD + in aqueous solutions was examined polarographically and by macroscale electrolysis. The mechanism of the reduction process was formulated. The surface activities of Nm 7 GD + and NGD + were confirmed by ac polarography. Intramolecular interactions of Nm 7 GD + at pH 5.0 and 8.93 were studied by fluorescence measurements, and the thermodynamic parameters for this process were determined.

Studies on Association of mRNA Cap-analogues with a synthetic Dodecapeptide DGIEPMWEDEKN

Abstract 1H NMR and fluorescence were applied to a study of interactions between dinucleotide analogues of mRNA cap and a peptide DGIEPMWEDEKN. Structures of the m7G-Trp complexes were determined by means of the ring current anisotropy theory, and their features were refered to the known X-ray structure of the eIF4E-m7GDP associate.