Biomedical subjects

G Fronza

Publications and source records attributed to G Fronza.

At least 55 records · Page 3Linked to original sources

PubMed1996

Genetic alterations in astrocytic tumors: hints to a molecular epidemiological interpretation.

General incidence of brain tumors has significantly increased over the past two decades. Although the aetiology of this increase has not been determined, increased life span and improved diagnosis methods can partially be responsible for it. Starting from the epidemiological data on risk factors, we reviewed the molecular events known to be involved in the genesis and progression of the most common brain neoplasm in adult, namely astrocytoma. Alterations in different genes, encoding key regulatory elements in the cell cycle control, were reviewed. In light of the molecular epidemiological notion that carcinogens leave fingerprints, point mutations at the p53 locus from a panel of astrocytic tumor patients from all over the world were analysed. The results of this analysis suggest that the majority of astrocytomas may have a spontaneous origin. In particular, the kind of mutational events observed suggests a major role for mutagenic events occurring at CpG sites. In order to yield valid conclusions on the potential role of environmental mutagenic factors, well designed molecular epidemiological studies on populations clearly showing a higher relative risk of developing brain tumors are needed.

Adult↗

PubMed1996

DNA adducts and chronic degenerative disease. Pathogenetic relevance and implications in preventive medicine.

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

Animals↗

PubMed1995

Mutational fingerprint induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea in mammalian cells.

Using the pZ189 shuttle vector approach, we determined two chloroethyl-cyclohexyl-nitrosourea (CCNU)-induced mutation spectra (3 and 6 mM) in African green monkey kidney cells (CV1). One hundred and twenty-one independent clones (101 CCNU induced, 45 at 3 mM and 56 at 6 mM; 20 spontaneous) showing functional inactivation of the supF gene were analyzed. One hundred and five plasmids (91 CCNU induced, 41 at 3 mM and 50 at 6 mM; 14 spontaneous), showing no large deletion/rearrangements, were sequenced. Ninety mutants (81 CCNU induced and 9 spontaneous) showed at least one mutation in the supF region. The analysis of the 122 CCNU-induced mutations (56 and 66 at 3 and 6 mM, respectively) revealed that: (a) the majority of the mutations were GC-targeted base pair substitutions; (b) AT-targeted mutations were significantly more frequent in the CCNU-induced (6 mM) than in the spontaneous mutational spectrum (P < 0.0006, Fisher's exact test); (c) mutational spectra obtained at 3 and 6 mM CCNU were significantly different (P < 0.008); (d) induced mutations were nonrandomly located in both spectra and generated either a common hot spot (position 123, 5'-GGG-3') or hot spots exclusive for each CCNU concentration (3 mM: position 159, 5'-AGG-3'; 6 mM: position 109, 5'-GGG-3'); (e) the occurrence of GC-->AT transitions was significantly different as a function of CCNU concentration (P < 0.02, Fisher's exact test), the mutated G being almost exclusively preceded by a purine (5'Pu G) at 6 mM and by either Pu or Py at 3 mM; and (f) by applying Calladine's rules, we found that sequences encompassing the three CCNU hot spots shared identical helix parameters for no more than 2 bp steps 5' (or 3 bp steps 3') to the mutated G. Our results are consistent with the hypothesis that O6-alkylguanine is responsible, either directly or indirectly, for the majority of GC-targeted mutations, while O4-alkylthymine and/or N3-alkyladenine are probably responsible for AT-targeted mutations. The results suggest also that, in CV1 cells, the efficiency of the repair mechanism(s) involved in the removal of O6-alkylguanine is influenced by the DNA sequence context. All of these factors determine the CCNU mutational fingerprint. CCNU has been implicated in the induction of therapy-related leukemias.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Cancer biomarkers in human atherosclerotic lesions: no evidence of p53 involvement.

In order to assess similarities between the atherogenic and the carcinogenic processes, we investigated whether the p53 tumor suppressor gene, the most commonly altered gene in human cancer, may be also involved in human atherosclerotic lesions. The medium layers of abdominal aorta fragments taken at surgery from 32 patients were subjected to immunohistochemical analysis, using either monoclonal (Pab 1801) or polyclonal (CM-1) antibodies, and to molecular analysis by the PCR-based denaturing gradient gel electrophoresis approach. The results obtained indicated that p53 mutations are not involved in the pathogenesis of atherosclerotic lesions, and that no accumulation of the wild-type protein occurs in smooth muscle cells of these lesions. A polymorphism characterized by an AT to GC transition at codon 213 (CGA --> CGG) causing no aminoacid substitution (Arg --> Arg) was detected in the 10.5% of the examined patients. Our negative findings do not support the hypothesis that the atherosclerotic plaques may be pathogenetically akin to benign tumors yet they are not in contrast with this theory, since in most cases p53 is involved in advanced stages of the carcinogenesis process.

Aged↗

PubMed1995

A nuclear magnetic resonance method for the determination of the purity of commercial dequalinium chloride.

4-amino-1-(10-4'-quinaldinylaminodecyl)quinaldinium chloride (3) has been identified through nuclear magnetic resonance (NMR) studies as one of the impurities of a commercial sample of dequalinium chloride (CAS 522-51-0). The NMR technique is proposed as a rapid method for the recognition and quantification of the impurities in dequalinium chloride samples.

Chromatography, High Pressure Liquid↗

PubMed1994

Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector DNA transfected into monkey cells.

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. We recently determined the mutation spectrum induced by the ultimate metabolite of 4NQO, acetoxy-4-aminoquinolone 1-oxide in the M13lacZ'/E. coli lacZ delta M15 alpha-complementation assay. Our data suggested that dGuo-C8-AQO induces (per se or via AP sites) G to Pyr transversions. Here we report our study on 4NQO mutagenesis in monkey cells. 4NQO lesions were induced in vitro on a single-stranded (ss) DNA shuttle vector carrying the supF tRNA gene. This vector was able to replicate both in mammalian cells and in bacteria. The mutations induced in monkey cells were screened by the white/blue beta-galactosidase activity assay in E. coli. We took advantage of the peculiar feature of ss supF DNA in which the extent of secondary structure may be a function of the temperature, with the dependence of the 4NQO-specific adduct spectrum on DNA secondary structure. We reasoned that mutational spectra derived from damage induced in the presence (20 degrees C) or absence (70 degrees C) of DNA secondary structure should be different. The result of sequencing a total of 89 induced and spontaneous mutants confirmed that the spectra are statistically different. These data suggest that the two 4NQO guanine adducts may induce different mutations.

4-Nitroquinoline-1-oxide↗

PubMed1994

The ultimate carcinogen of 4-nitroquinoline 1-oxide does not react with Z-DNA and hyperreacts with B-Z junctions.

DNA secondary and tertiary structures are known to affect the reaction between the double helix and several damaging agents. We have previously shown that the tertiary structure of DNA influences the reactivity of 4-acetoxyaminoquinoline 1-oxide (Ac-4-HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide (4-NQO), being more reactive with naturally supercoiled DNA than with relaxed DNA. The relative proportion of the three main stable adducts and of an unstable adduct, that resulted in strand scission and/or AP sites, was also affected by the degree of supercoiling of plasmid DNA. In this study we examined the influence of Z-DNA structure on the reactivity of Ac-4-HAQO by mapping the distribution of the two main Ac-4-HAQO adducts, C8-guanine and N2-guanine, along a (dC-dG)16 sequence inserted at the BamHI site of pBR322 plasmid DNA. This insert adopted the left-handed Z and right-handed B structure depending on the superhelical density of the plasmid. Sites of C8-guanine adduct formation were determined by hot piperidine cleavage of Ac-4-HAQO modified DNA, while N2-guanine adducts were mapped by the arrest of the 3'-5' exonuclease activity of T4 DNA polymerase. The results showed that Ac-4-HAQO did not react with guanine residues when the (dC-dG)16 sequence was in Z conformation, while hyperreactivity at the B-Z junction was observed. These results indicate that Ac-4-HAQO can probe the polymorphism of DNA at the nucleotide level.

4-Nitroquinoline-1-oxide↗

PubMed1994

Defective splicing induced by 4NQO in the hamster hprt gene.

The molecular analysis of mutations affecting mRNA processing may contribute to a better understanding of the splicing mechanism through the identification of genomic sequences necessary for the recognition of splice sites. In this paper we report the sequence analysis of 14 splice mutants induced by 4-nitroquinoline 1-oxide (4NQO) at the hamster hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus. We show that mutations at the 3' acceptor splice site or at the first or fifth base of the 5' donor splice site are responsible for exon skipping. In addition, mutations in exon sequences also determine the skipping of one or more exons. Our data indicate that point mutations in intron regions at either side of an internal exon may induce the skipping of the same exon, supporting a model where the exon is the unit of early spliceosome assembly. Furthermore, they suggest that the splicing of hprt mRNA precursors may proceed through a clustering of exons 2, 3 and 4 which are then spliced in a concerted way.

4-Nitroquinoline-1-oxide↗

PubMed1994

Synthesis, absolute configuration, conformational analysis and binding affinity properties of enantiomeric forms of DAU 5750, a novel M1-M3 muscarinic receptor antagonist.

Both the enantiomeric forms of DAU 5750, a novel muscarinic receptor antagonist, have been synthesized in order to assess the relevance of configurational/conformational features for high affinity binding to muscarinic receptor subtypes. The attribution of absolute stereochemistry and conformational analysis by means of molecular modelling and NMR techniques are also reported.

Chymotrypsin↗

PubMed1994

Analysis of 4-nitroquinoline-1-oxide induced mutations at the hprt locus in mammalian cells: possible involvement of preferential DNA repair.

Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO) at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5) Chinese hamster ovary cells were determined to investigate the effect of DNA repair on the nature of induced mutations. The UV5 cell line is three times more sensitive to 4NQO than the AA8 parental cell line. In UV5 cells, the dGuo-N2-AQO adduct, which is considered to be the most toxic and mutagenic adduct in Escherichia coli, is poorly repaired. The molecular nature of 30 hprt mutants isolated from AA8 and 20 isolated from UV5 cells was determined by sequence analysis of in vitro amplified hprt cDNA. Both similarities and differences emerged. In both cell lines we found that (i) 4NQO is basically a base substitution mutagen acting almost exclusively at G residues and (ii) G transversions are prevalent over G transitions in both cell lines, independently from the ability to repair dGuo-N2-AQO. A high proportion (13/25) of splice mutations was observed in AA8 cells, statistically different (P < 0.04, Fisher's exact test) from the incidence of splice mutants in UV5 cells (4/20). In AA8 mutants, all but two of the point mutations were due to lesions localized on the non-transcribed strand, suggesting preferential repair of the transcribed strand. Compared with AA8, the proportion of mutants due to lesions present on the transcribed strand was higher in UV5 cells, as expected if a preferential repair mechanism was impaired in the sensitive cell line. Our data are consistent with the molecular defect in DNA repair recently characterized in UV5.

4-Nitroquinoline-1-oxide↗

PubMed1993

Non-peptide ligands for opioid receptors. Design of kappa-specific agonists.

A series of phenyl carboxyl esters 5a-d derived from N-(cyclopropylmethyl)normetazocine was synthesized and evaluated for its selectivity at mu, kappa, and delta opioid receptors. Compound 5a, although 43 times less potent than the reference compound U50488, was specific for kappa receptors, having no detectable affinity for either mu or delta receptors. Greater binding affinity was seen with the diastereoisomer having the 1'R,2'S stereochemistry in the cyclopropyl ring of the nitrogen substituent, which was only 12 times less active than U50488. Antinociceptive activity in the mouse tail flick was only slightly lower than that of U50488 (ED50 = 7.66 vs 4.52 mg/kg). Naloxone fully prevented antinociception induced by (1'R,2'S)-5a at the doses of 2.0 mg/kg. Compound (1'R,2'S)-5a is one of the most kappa-selective non-peptide compounds reported to date. The implications of these results in terms of requirements for kappa ligands are discussed.

Animals↗

PubMed1992

The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions.

4-Nitroquinoline-1-oxide is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. In ds or ss damaged DNA the ratio C8/N2 adducts is 1:2 and 8-10:1, respectively. In bacteria and yeast 4NQO has been shown to be a base substitution mutagen acting at G residues inducing mainly G to A transitions. We determined the mutational spectrum induced by the 4NQO metabolite, acetoxy-4-aminoquinoline 1-oxide, in the M13lacZ'/E. coli lacZ delta M15 alpha complementation assay using ssDNA. Among 68 Ac-4HAQO induced mutants, G to Pyr transversion was the most frequent base substitution observed. By comparison with dsDNA based systems, our data suggest that dGuo-C8-AQO induces G to Pyr transversions. A mechanism to explain how this lesion may induce transversions is proposed.

4-Nitroquinoline-1-oxide↗

PubMed1992

Identification of the major degradation products of 4-methoxy-2-(3-phenyl-2-propynyl)phenol formed by exposure to air and light.

Exposure of 4-methoxy-2-(3-phenyl-2-propynyl)phenol (CO/1828) to air and light (accelerated by temperature) yields 1-(2-hydroxy-5- methoxyphenyl)-3-phenylpropynone as the major degradation product. With extraction, this product rapidly degrades to 5-methoxyaurone and 6-methoxyflavone. In addition, a mixture of dimeric and heterodimeric compounds that are not fully identified was observed. These results indicate the formation of a reactive ortho-quinone methide as an unstable intermediate. This hypothesis is supported by evidence that the aurone slowly isomerizes into the flavone in control samples. Identification of compounds was accomplished with mass spectrometry, nuclear magnetic resonance spectrometry, UV-high-performance liquid chromatography, and comparison with authentic samples.

Administration, Topical↗

PubMed1992

Proton nuclear magnetic resonance spectroscopy studies of the inclusion complex of piroxicam with beta-cyclodextrin.

Proton nuclear magnetic resonance spectroscopy showed that piroxicam sodium salt forms an inclusion complex with beta-cyclodextrin in aqueous solution. The 1:1 stoichiometry of the complex was determined by the continuous variation method. Significant nuclear Overhauser effects were observed between the inner protons of beta-cyclodextrin and protons of both the aromatic rings of piroxicam sodium salt, a result indicating that two isomeric 1:1 complexes must be present in solution within the range of concentrations investigated. The overall association constant was 113 M at 298 K. At concentrations less than 1.0 x 10(-3) M, the complex is completely dissociated.

Chemical Phenomena↗

PubMed1992

Single stranded DNA-vectors for analyzing processing of DNA damage induced by 4-nitroquinoline-1-oxide in prokaryotes and eukaryotes.

Previous studies indicate that single stranded DNA vectors could be used in different organisms to study mutagenesis induced by DNA damaging agents. We applied this approach to study mutagenesis induced by 4NQO lesions. The use of ssDNA, on which the ultimate metabolite of 4NQO (Ac-4HAQO) induces mainly C8-guanine adducts, allowed us to find a correlation between G-transversions and the dGuo-C8-AQO adduct. This correlation was established in two independent assay-systems, based on prokaryotic and eukaryotic cells.

4-Nitroquinoline-1-oxide↗

PubMed1992

Multi-system approach to study mutagenesis induced by chemical carcinogens.

To understand molecular mechanisms of the mutation fixation process induced by a mutagen and carcinogen, a multi-system approach is suggested to reduce the probability that the results are biased by the assay used. In this light we described our different approaches to answer basic questions on the mutagenesis induced by the chemical carcinogen 4-Nitroquinoline-1-oxide. We determined mutations at the molecular level in three experimental systems: a) in prokaryotes (ss M13mp19 lacZ'/E. coli F'lacZ delta M15); b) in eukaryotes (i) ss and ds pZ189 supF/CV1-P/E.coli lacZam and (ii) HPRT in CHO cells with different repair capacity. We think this type of approach can be used to study the genetic effects of new cancer drugs for which the molecular mechanisms of action at the molecular level are still not well understood. We think to apply the know-how to study mutational spectra in tumor derived tumor suppressor genes.

4-Nitroquinoline-1-oxide↗

PubMed1991

Three dimensional structure of GD1b and GD1b-monolactone gangliosides in dimethylsulphoxide: a nuclear Overhauser effect investigation supported by molecular dynamics calculations.

A comparative study on the conformational features of the oligosaccharide moiety of GD1b and GD1b lactone gangliosides, in dimethylsulphoxide, has been carried out by nuclear Overhauser effect investigation; the experimental interresidue contacts have been used for restrained molecular mechanics and dynamics calculations. For GD1b, the tetrasaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2 ----8)-alpha-Neu5Ac-(2----3)]-beta-Gal has a circular arrangement leaving a highly hydrophobic region with seven hydrogens pointing towards the center. At one side of this region the three electron rich groups GalNAc--NH, external Neu5Ac--OH4 and internal Neu5Ac--COO- are grouped together; at the other side five polar groups (four hydroxy groups and the external Neu5Ac carboxylate) define a large annular hydrophilic region. The external Neu5Ac is close to the external Gal residue, and the external Neu5Ac--COO- is within van der Waals contact with the inner Neu5Ac-OH9 group. The beta-Gal-(1----3)-beta-GalNAc glycosidic linkage shows a high degree of freedom. For GD1b-L, the trisaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2----3)]-beta-Gal is disposed to forming rigid partially circular arrangement showing strong interresidue contacts between the inner Neu5Ac-H8 and both GalNAc-H1 and GalNAc-H5. The conformation of the lactone ring is the boat 9(A),2(B)B. The lactonization of the disialosyl residue induces a strong variation of the preexisting torsional glycosidic angles phi and psi, leaving the external Neu5Ac far from the external Gal. In both GD1b and GD1b lactone gangliosides, the conformation of the sialic acid side chain is the same as that of the free sialic acid in which the H7 is trans to H8 and gauche to H6, thus indicating that the presence of glycosidic and/or ester linkages does not affect the conformational properties of sialic acid. Both GD1b and GD1b lactone containing sialic acid carboxylate anion(s) or undissociated carboxyl group(s) show the same three dimensional structure, indicating that the presence of charges does not affect the intrinsic conformational features of gangliosides.

Carbohydrate Conformation↗

PubMed1991

Relationship between DNA topoisomerase I activity and DNA synthesis in cultured hepatocytes: effects of orotic acid.

Previous studies have demonstrated that DNA topoisomerase I activity can be closely related to DNA replication and active transcription in different experimental models. This relationship was further investigated by studying the time course of DNA topoisomerase I activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. DNA topoisomerase I activity was assayed quantifying ATP-independent relaxation of a negatively supercoiled plasmid, substrate for the enzyme. Incubation of cellular extracts was carried out at 30 degrees C for 15'. Products were electrophoresed and analyzed by densitometry. DNA synthesis was measured as [3H]thymidine incorporation in the hepatocytes, after a 2 hrs pulse labelling. An increase in DNA topoisomerase I activity was observed early after epidermal growth factor addition, before the onset of DNA synthesis. Concomitant administration of orotic acid, which has recently shown mitoinhibitory effect, abolished the EGF-induced activity as well as DNA synthesis. Taken together these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by DNA topoisomerase I with a precise time schedule. Orotic acid administration abolishes this response and causes an overall loss in DNA topoisomerase I activity.

Animals↗