Biomedical subjects

J Retèl

Publications and source records attributed to J Retèl.

At least 37 records · Page 2Linked to original sources

PubMed1991

A circular dichroism study on the conformation of d(CGT) modified with N-acetyl-2-aminofluorene or 2-aminofluorene.

The trinucleotide d(CGT) was modified by covalent binding of the carcinogen N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) at the C8 position of the guanine base. The conformations of d(CGT)-AAF and -AF were studied by comparing the absorption and circular dichroism properties with those of dCMP + dGMP-AAF or -AF + dTMP in a molar ratio of 1:1:1 and AAF- and AF-containing dGMP. For both AAF- and AF-d(CGT) complexes the results show significant stacking interactions between the fluorene residue and the base(s) and are discussed in terms of the conformation of d(CGT)-AAF and -AF. In d(CGT)-AF we observe a clear interaction between AF and thymine, whereas the C-G stack is still intact. In the case of d(CGT)-AAF the C-G stack is weakened and the glycosidic rotation angle of dGuo-C8-AAF is most probably syn. The specific fluorene-base interactions persist at elevated temperatures. The carcinogen-base interactions are stronger in the AAF-carrying d(CGT) than in the case of the deacetylated complex. This is consistent with the higher mobility of the AF-adduct and its conformationally heterogeneous appearance in DNA.

2-Acetylaminofluorene↗

PubMed1991

Mutational specificity of gamma-rays differs for the same target in plasmid DNA and double-stranded (RF) M13 bacteriophage DNA.

In the lacZ alpha gene of a pUC plasmid a 144 bp insert was cloned as target for mutagenesis. Irradiation of the plasmid in a diluted aqueous solution by 60Co gamma-rays under oxic conditions leads to a very specific mutation spectrum. The predominant type of mutation was a C/G to A/T transversion (29 out of 47 mutants) whereas C/G to G/C transversions were found 7 times and C/G to T/A transitions 10 times. Only one frameshift could be observed which was a deletion of an A/T base pair. The mutations were not randomly distributed along the mutation target but show a strong preference for a certain DNA sequence in which two thirds of the mutations were scored. In this DNA area a hotspot (24 of the 47 mutants) for mutagenesis was located and within 6 bp next to this hotspot another seven mutations were scored. The mutation spectrum in the same mutation target as part of double-stranded (RF) M13 phage was published before. In both systems the mutational hotspot is located at the same site, but the predominant type of mutation is different. In the M13 system the C/G to G/C transversion was the most important event.

Bacteriophages↗

PubMed1990

Role of the semi-quinone free radical of the anti-tumour agent etoposide (VP-16-213) in the inactivation of single- and double-stranded phi X174 DNA.

The mechanism of action of the anti-tumour agent etoposide (VP-16-213) could involve its bioactivation to metabolites which can damage DNA. Active metabolites of etoposide, generated in vitro, are the 3',4'-dihydroxy-derivative (catechol) and its oxidation product, the ortho-quinone. The conversion of the catechol into the ortho-quinone (and vice versa) proceeds via formation of a semi-quinone free radical. We investigated the role of this radical species in the inactivation of biologically active single- (ss) and double-stranded (RF) phi X174 DNA. Since the formation of semi-quinone free radicals from the ortho-quinone of etoposide is pH dependent, experiments were performed, in which the ortho-quinone was incubated at pH 4, 7.4 and greater than or equal to 9. ESR measurements showed no formation of radical species from the ortho-quinone at pH 4, but an increased rate of generation of the primary semi-quinone free radical at pH values 7.4 to 10; at still higher pH values a secondary semi-quinone free radical was produced. HPLC analyses demonstrated chemical stability of the ortho-quinone at pH 4, but an accelerated decay was observed when the pH was elevated from 7.4 to 9, with its concomitant conversion into more polar components and into the catechol of etoposide. Ss phi X174 DNA, exposed to the ortho-quinone, was inactivated at an increasing rate at pH values increasing from 4 to 7.4 and subsequently to 9. RF phi X174 DNA was only significantly inactivated in incubations with the ortho-quinone at pH 4, not at pH values 7.4 and 9. From these data it is concluded that the primary semi-quinone free radical of etoposide may to a great extent be responsible for the ortho-quinone-induced ss phi X174 DNA inactivation, but that this radical species is not lethal towards RF phi X174 DNA.

Bacteriophage phi X 174↗

PubMed1990

The fluorescence emission properties of dGuo-C8-AAF, dGuo-C8-AF and the imidazole ring-opened products of dGuo-C8-AF indicate a different dynamic structure for the various compounds.

The reactive metabolites of the carcinogenic N-acetyl-2-aminofluorene (AAF) form adducts with the guanine base of DNA. The fluorescence emission characteristics of N-(deoxy-guanosin-8-yl)-N-acetyl-2-aminofluorene (dGuo-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dGuo-C8-AF) and the two N7 = C8 imidazole ring-opened products of dGuo-C8-AF (ro-dGuo-C8-AF I + II) were investigated and related to their conformational properties. The dGuo-C8-AF adduct (phi F congruent to 4-5 X 10(-4) shows a broad and structureless emission band, which is attributed to the formation of an excited-state complex. In contrast, the emission spectra of dGuo-C8-AAF (phi F congruent to 1.10(-4] and both ro-dGuo-C8-AF compounds [phi F(ro-dGuo-C8-AF I) congruent to 4.10(-3); phi F(ro-dGuo-C8-AF II) congruent to 4.10(-4)] are narrow. This indicates that dGuo-C8-AAF and ro-dGuo-C8-AF I + II do not decay into an exciplex as occurs in dGuo-C8-AF. The spectroscopic features are discussed in terms of the differences in the dynamic structure of the various compounds.

2-Acetylaminofluorene↗

PubMed1990

Effects of oxygen radical scavengers on the inactivation of SS phi X174 DNA by the semi-quinone free radical of the antitumor agent etoposide.

We have studied the effects of oxygen radical scavengers on the inactivation of ss phi X174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH greater than or equal to 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss phi X174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO, the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH greater than or equal to 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH. trapping but from trapping of O2-(.). DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH greater than or equal to 7.4. Potassium superoxide also stimulated phi X174 DNA inactivation by the ortho-quinone at pH less than or equal to 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the ortho-quinone of etoposide, thus indirectly influencing DNA inactivation.

Bacteriophage phi X 174↗

PubMed1989

Mutations induced by 60Co gamma-irradiation in double-stranded M13 bacteriophage DNA in nitrous-oxide saturated solutions are characterized by a high specificity.

Irradiation of double-stranded M13 mp10 DNA in a diluted aqueous solution under N2O leads to a very specific mutation spectrum. Fifteen of 28 mutations induced in a 144 base pair (bp) target are C/G to G/C transversions, the other five bp substitutions are C/G to A/T transversions. Six mutations were single bp deletions, one is a large deletion of 180 bp and one is a 10 bp duplication which is probably from spontaneous origin. The mutations are not randomly distributed throughout the 144 bp mutation target but concentrated around two sites. The differences and similarities with the radiation-induced mutation spectrum previously obtained under oxygen are discussed.

Base Sequence↗

PubMed1989

Excited state properties of the N-(deoxyguanosin-8-yl)-2-aminofluorene adducts.

The spectroscopic characteristics of adducts derived from the covalent binding of the carcinogen 2-aminofluorene to the C8 position of deoxyguanosine [N-(deoxyguanosin-8-yl)-2-amino-fluorene, dGuo-C8-AF], and from an adduct of similar structure formed with the synthetic polynucleotide poly(dG-dC).poly(dG-dC), were investigated. At 77 K both adducts are characterized by well-defined and rather narrow fluorescence emission spectra with maxima at 370 and 390 nm characteristic of the aromatic, monomolecular 2-aminofluorene (AF) residue. In contrast, at room temperature, the fluorescence is characterized by a broad, structureless emission band with a maximum at 460 nm in aqueous mixtures, shifting to 415 nm in solvents of lower polarity (100% propanol); the maxima are located at intermediate wavelengths in solutions of different propanol/water compositions, and this emission is attributed to an excited state complex (exciplex). The fluorescence quantum yield decreases when either the solvent polarity or the temperature are increased, varying from 5.4% (100% propanol) to 0.04-0.05% (100% H2O). The fluorescence decay profiles of dGuo-C8-AF adducts (measured at the National Synchrotron Light Source facility at the Brookhaven National Laboratory) can be roughly, but not exactly, modeled in terms of two exponential decay components in the range of about 0.3-1.0 ns with the propanol concentration greater than 60%; at lower propanol concentrations, a single short lifetime is observed and in 100% water solutions its value is 0.08 ns. The shorter lifetime, favored in solvent mixtures of higher polarities, is attributed to an exciplex with significant charge-transfer character.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Acetylaminofluorene↗

PubMed1988

Mechanism of action of antitumor drug etoposide: a review.

Metabolism studies of the antitumor drug etoposide show the formation of metabolites in the lactone ring, which are probably not important for the drug's mechanism of action, and oxidative transformations in the dimethoxyphenol ring (E ring), which lead to products that can cause DNA damage and may play a role in the drug's mechanism of action. The cytotoxicity of etoposide is caused by the induction of DNA damage. The occurrence of the DNA lesions can be explained by the capacity of the drug to interfere with the scission-reunion reaction of mammalian topoisomerase II by stabilizing a cleavable complex.

Biotransformation↗

PubMed1988

Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded phi X174 DNA.

We have studied the effects of the recently reported two new metabolites of the antitumor agent VP-16-213, the ortho-dihydroxy derivative or catechol and the ortho-quinone, on the biological activity of single-stranded and double-stranded phi X174 DNA, the binding of the metabolites to calf thymus DNA and the conversion of the catechol into the ortho-quinone. Evidence was obtained for the oxidation of the catechol into the ortho-quinone and for the fact that the ortho-quinone is the metabolite of VP-16-213 responsible for its binding to rat liver microsomal proteins. The catechol and ortho-quinone of VP-16-213 were found to bind 7-9 times more strongly to calf thymus DNA than VP-16-213 itself. In contrast to the parent compound VP-16-213, the catechol as well as the ortho-quinone inactivated both single-stranded (ss) and double-stranded (RF) biologically active phi X174 DNA. The mean T37-values for inactivation of ss and RF phi X174 DNA by 2.2 x 10(-4)M catechol at 37 degrees and pH 7.4 were 96 and 640 min, respectively. Reduction of the ortho-quinone by NADPH cytochrome P-450 reductase resulted in formation of the catechol. The system ortho-quinone/NADPH cytochrome P-450 reductase inactivated ss phi X174 DNA with a mean T37-value of 454 min, and this inactivation was inhibited by DMSO. The mean T37-value for inactivation of ss phi X174 DNA by 1.8 x 10(-4) M ortho-quinone at 37 degrees and pH 4.0 was 24 min. The chemical stability of the ortho-quinone and the extent of inactivation of ss phi X174 DNA by the ortho-quinone were both pH-dependent: at higher pH the ortho-quinone was less stable and gave less inactivation of DNA. The aqueous decomposition product(s) of the ortho-quinone formed at pH 7.4 inactivated ss phi X174 DNA with a mean T37-value of 175 min. The rate of inactivation of RF phi X174 DNA by the ortho-quinone at pH 4.0 was twice as low as the rate of inactivation of ss phi X174 DNA: T37 = 49 min. When using excision repair deficient E. coli mutants (uvrA- or uvrC-), a higher inactivation of RF phi X174 DNA was found: T37 = 29 min for uvrA- E. coli, indicating that a part of the DNA damage introduced by the incubation with ortho-quinone is removed by excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

60Co gamma-rays induce predominantly C/G to G/C transversions in double-stranded M13 DNA.

Upon irradiation with gamma rays of an oxygenated aqueous solution of double-stranded M13 DNA, a very specific mutation spectrum was found with respect to both the type and the positions in the DNA sequence. Of the 23 mutations, which were sequenced, 16 represent a C/G to G/C transversion. A C/G to T/A transition was found once and a G/C to T/A transversion twice. The remaining 4 mutations are frameshifts, 2 are identical and formed by the insertion of a G/C basepair; the other 2 mutations are due to a duplication of 10 basepairs situated at different positions but with a remarkable homology in base sequence. Fourteen mutations, including the 2 duplications are found in the neighbourhood of a TGCT/ACGA sequence.

Base Sequence↗

PubMed1988

Structure-bioactivation relationship of a series of podophyllotoxin derivatives.

With the aim of elucidating the structural requirements for O-demethylation of the antitumor agent VP-16-213 by cytochrome P-450, the binding of a series of podophyllotoxin derivatives to rat liver microsomal cytochrome P-450 was studied. The examined podophyllotoxin derivatives were: VP-16-213, VM-26, podophyllotoxin, 4'-demethylepipodophyllotoxin (the aglycone of VP-16-213 and VM-26) and 3,5-dimethoxy-4-hydroxytoluene (a model compound for the E-ring of VP-16-213). The binding to phenobarbital (Pb)-induced microsomes was more extensive than that to 3-methylcholanthrene (3-MC)-induced microsomes. Experiments on the binding to cytochrome P-450 in Pb-induced microsomes led to the following findings: (a) the presence of the polycyclic skeleton is necessary for binding; (b) the presence of the sugar moiety gives a further extension of binding, and changes in the sugar moiety affect binding; (c) binding increases on elevation of hydrophobicity; (d) the E-ring itself does not bind. For binding to cytochrome P-450 in 3-MC-induced microsomes conclusions (a) and (d) appeared to hold true. For the O-demethylation of the podophyllotoxin derivatives containing the dimethoxyphenol ring by Pb- and 3-MC-induced microsomes, the following order was observed: VM-26 greater than VP-16-213 greater than aglycone much greater than E-ring. A similar sequence was observed for the cytotoxicity against Chinese hamster ovary cells.

Animals↗

PubMed1988

A spectroscopic study of the conformation of poly d(G-C).poly d(G-C) modified with the carcinogenic 2-aminofluorene.

The conformational properties of both the B- and Z-form helix of the 2-aminofluorene (AF) modified synthetic polynucleotide poly d(G-C).poly d(G-C) were extensively studied with a variety of optical techniques (UV absorbance, linear dichroism, circular dichroism and fluorescence spectroscopy). The spectroscopic results show, that: (i) the presence of the AF adduct in poly d(G-C).poly d(G-C) facilitates the conversion of the right-handed B-form polynucleotide to the left-handed Z-type poly d(G-C).poly d(G-C) duplex; (ii) in B- as well as in Z-form poly d(G-C).poly d(G-C)-AF the carcinogenic residue is situated in a specific and ordered complex with the DNA, in which the AF chromophore is significantly immobilized and the long axis of the fluorene ring system is positioned in a parallel mode to the DNA base planes; (iii) in B-type poly d(G-C).poly d(G-C)-AF the carcinogenic residue exhibits strong stacking interactions with the adjacent DNA bases, which are coupled to an important destabilization of the AF-containing B-duplex; (iv) in contrast to the B-form complex, the AF-containing Z-type DNA is highly stabilized and a remarkable reduction of the AF-base interactions is observed; (v) in Z-form poly d(G-C).poly d(G-C)-AF the AF chromophore resides at a medium-exposed position. The combined data support a conformational model, in which the planar AF is inserted in the B-type polynucleotide helix, while the carcinogenic residue in the Z-form poly d(G-C).poly d(G-C)-AF structure is placed in an outside position.

Carcinogens↗

PubMed1988

Semi-quinone formation from the catechol and ortho-quinone metabolites of the antitumor agent VP-16-213.

The catechol metabolite of the antitumor agent VP-16-213 and the ortho-quinone of VP-16-213--a secondary metabolite formed from the catechol--easily undergo auto-oxidation into a free radical at pH greater than or equal to 7.4. By elevation of the pH from 7.4 to 10, an increase in the production of the free radical was observed, which was accompanied by the formation of products with higher hydrophylicity than the catechol and ortho-quinone, as found by HPLC-analysis. The hyperfine structure of the free radical indicates that it is the semi-quinone radical of VP-16-213. At pH 12.5 a secondary radical is formed from the catechol and the ortho-quinone of VP-16-213 besides the semi-quinone radical. One-electron oxidation of the catechol with horseradish peroxidase/hydrogen peroxide resulted in the formation of the same radical as observed under alkaline conditions and subsequent oxidation to the ortho-quinone. If the ortho-quinone was incubated with NADPH cytochrome P-450 reductase, a free radical was detected by spin-trapping with POBN, but not without spin-trapping. Studies on inactivation of phi X174 DNA by the system ortho-quinone of VP-16-213/NADPH cytochrome P-450 reductase suggest that the semi-quinone radical may play a role in the process of inactivation of DNA.

Bacteriophage phi X 174↗

PubMed1987

Cytochrome P-450-mediated O-demethylation: a route in the metabolic activation of etoposide (VP-16-213).

The antitumor agent VP-16-213 is oxidatively O-demethylated by rat liver microsomes and purified rat liver microsomal cytochrome P-450. 3-Methylcholanthrene can quantitatively induce O-demethylation of VP-16-213. The Km and Vmax values for O-demethylation by noninduced, phenobarbital-, and 3-methylcholanthrene-induced rat liver microsomes were found to be 130, 600, and 160 microM and 8.5, 11.8, and 15.6 nmol H2CO/min X mg protein, respectively. Mass spectrometric comparison of the product of O-demethylation of VP-16-213 with the synthetic metabolite resulted in identification of the orthodihydroxy derivative. In studies on the biological activity of the orthodihydroxy derivative, it was found to inactivate single- and double-stranded phiX174 DNA, to bind to calf thymus DNA and to be highly toxic against chinese hamster ovary cells.

Animals↗

PubMed1987

An optical study of the conformation of the aminofluorene-DNA complex.

Calf thymus DNA was modified with 2-aminofluorene (AF) to different extents by treatment with N-hydroxy-2-aminofluorene. The AF-modified DNAs together with free AF, the AF-modified guanine (Gua-C8-AF) and the AF-modified deoxyguanosine (dGuo-C8-AF) were subsequently studied by u.v. absorbance, linear dichroism and fluorescence spectroscopy. The emission and absorption properties of double-stranded DNA-AF and single-stranded DNA-AF closely resemble those of dGuo-C8-AF. The emission spectra of these three compounds show a broad, red-shifted emission, characteristic for exciplex formation. The linear dichroism and circular dichroism spectra of double-stranded DNA-AF show that the AF moiety forms a well-defined, regular structure. The dichroic ratio in the 310-340 nm region is constant, which indicates the presence of only one type of adduct. The long-wavelength transition moment of this adduct makes an angle of 72-74 degrees with the DNA helix axis. The binding of AF to double-stranded is DNA is accompanied by a destabilization of the DNA helix structure, a strong quenching of the AF emission quantum yield, intense AF circular dichroism and an apparent immobilization of the dGuo-C8-AF complex. In single-stranded DNA-AF, the AF conformation appears more random, although the interactions between AF and the surrounding bases persist. The strong interactions between AF and the surrounding bases which dominate the optical properties of the studied complexes, the significant destabilization of the DNA double helix after modification with AF, and the relatively small angle between AF and the base planes support a model in which the adduct is inserted into the DNA helix.

Carcinogens↗

PubMed1986

The biological activity of single-stranded phi X174 DNA, modified by N-hydroxy-2-aminofluorene, is inhibited by guanine imidazole ring-opening of the major, non-lethal aminofluorene-DNA adduct.

The major aminofluorene-DNA derivative, found in the liver of rats after administration of the hepatocarcinogen N-acetyl-2-aminofluorene and identified as N-(deoxyguanosin-8-yl)-2-aminofluorene (dGuo-C8-AF), was introduced in different amounts in single-stranded phi X174 DNA by reacting the DNA with tritium labeled N-hydroxy-2-aminofluorene. The modified DNA was subsequently incubated in 0.1 M NaOH at 37 degrees C for increasing periods of time to convert the dGuo-C8-AF residues into their guanine imidazole ring-opened forms. The degree of conversion was determined by measuring the amount of residual N-(guanin-8-yl)-2-aminofluorene in trifluoroacetic acid hydrolyzates of the alkali-treated DNA by h.p.l.c. In addition, the effect of ring opening on the biological activity of the DNA was monitored by transfecting the DNA to Escherichia coli wild-type spheroplasts. The results indicate that the major aminofluorene-DNA adduct formed initially, which contributes little to inactivation, becomes lethal when its guanine imidazole ring is opened.

Bacteriophage phi X 174↗

PubMed1985

Inactivation of phi X174 DNA by the ortho-quinone derivative or its reduction product of the antitumor agent VP 16-213.

Biologically active phi X174 DNA is inactivated by the ortho-quinone derivative of the antitumor agent VP 16-213, but not by VP 16-213 itself, VP 16-213 phenoxy radical or aqueous decomposition product(s) of the ortho-quinone. Reduction of the ortho-quinone by cytochrome P-450 reductase and NADPH results in deactivation of the ortho-quinone towards anti-phi X174 DNA activity. However, compared with the parent compound VP 16-213, reduction of the ortho-quinone results in substantial damage towards DNA.

Bacteriophage phi X 174↗

PubMed1985

Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach.

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.

Bacteriophage phi X 174↗