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P Degan

Publications and source records attributed to P Degan.

At least 37 records · Page 2Linked to original sources
PubMed1994

Genotoxic effects of the carbamate insecticide methomyl. I. In vitro studies with pure compound and the technical formulation "Lannate 25".

The carbamate insecticide methomyl and the methomyl-containing technical formulation "Lannate 25" were tested on whole blood human lymphocyte cultures. Both products induced dose-dependent increases in chromosome aberrations and micronuclei. Lannate 25 induced DNA damage as measured by the alkaline elution assay and hydroxylation of guanine at the C8 position. Sister chromatid exchanges were not increased significantly with either product. Overall, the technical formulation was more active than the pure compound, when compared at similar concentrations of active principle. Moreover, a different ratio of CREST-positive/CREST-negative micronuclei was observed with the two products, pure methomyl being relatively more active than Lannate 25 in the induction of CREST-positive micronuclei. On the basis of these results, previous evaluations of methomyl as a nongenotoxic compound should be reconsidered.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Genotoxic effects of the carbamate insecticide, methyomyl. II. In vivo studies with pure compound and the technical formulation, "Lannate 25".

The carbamate insecticide, methomyl, and the methomyl-containing technical formulation, "Lannate 25", were tested for the induction of DNA damage in vivo. Swiss CD1 mice were treated intraperitoneally with test substances and the following tests were performed: alkaline elution of liver and kidney DNA, 8-hydroxyguanosine detection in liver DNA, and 32P-postlabelling analysis of DNA adducts in liver DNA. The clastogenic activity of the two pesticide preparations was also evaluated as micronucleus frequency in bone marrow. No DNA adducts were detected in liver DNA of mice treated with pure methomyl, while a dose-related increase in DNA adducts was found in Lannate 25-treated animals. All other tests were positive with both methomyl and Lannate 25. A summary of genotoxic activity of methomyl is also presented. The hypothesis that the observed genotoxic effects of methomyl are induced indirectly, through formation of active oxygen species, is discussed.

Animals↗
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

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↗
PubMed1992

Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column.

An immunoaffinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures of Escherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG;8-hydroxy-2'-deoxyguanosine), a bio-marker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8-dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 10(5) oxidative lesions to DNA being formed and excised in an average rat cell per day.

Animals↗
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

Immunoaffinity isolation of urinary 8-hydroxy-2'-deoxyguanosine and 8-hydroxyguanine and quantitation of 8-hydroxy-2'-deoxyguanosine in DNA by polyclonal antibodies.

An immunoaffinity column is described that facilitates the analysis of oxidative DNA damage. DNA adducts excised from DNA are excreted in urine and can be assayed as a measure of DNA damage in individuals. Polyclonal antibodies that recognize 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative damage to DNA, have been produced and their binding properties characterized. The antibodies, raised in rabbits following immunization with protein carrier-hapten conjugates prepared by covalently linking periodate-treated 8-hydroxyguanosine (oh8G) to bovine serum albumin (BSA) or casein, bind oh8dG with high affinity and selectivity, as measured by a competitive radioimmunoassay (RIA). Antibodies obtained from the rabbits immunized with the casein conjugate exhibited a binding affinity for oh8dG of 6.9 x 10(8) M-1. Studies on the relative binding affinities of these polyclonal antibodies for oh8dG, unmodified nucleosides, or derivatives of guanine indicate that the antibodies are suitable for the preparation of immunoaffinity columns that permit us to rapidly isolate oh8dG and 8-hydroxyguanine (oh8Gua) from urine. The high selectivity of the antibodies for oh8dG and oh8G reduces the amount of urinary contaminants previously observed in samples prepared by solid phase extraction, thus greatly facilitating the isolation of these damage products from urine. The relative binding affinity of these antibodies for oh8Gua and 2'-deoxyguanosine were approximately 7.6 x 10(3) and 7.4 x 10(4) fold lower respectively, than the binding affinity for oh8dG. The antibody can be used to quantitate oh8dG in enzymatic hydrolyzates of DNA with values comparable to those obtained by HPLC with electrochemical detection (HPLC-EC).

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1990

Time resolved fluoroimmunoassay of 7-methyl-2'-deoxyguanosine imidazole (ring open).

A solid-phase competitive time-resolved fluoroimmunoassay for 7-methyl-2'-deoxyguanosine imidazole (ring open) is described, based on highly specific hemocyanin carrier rabbit antibodies, modified with europium chelates. Eu3+ photoluminescence was detected in a novel micellar solution. The assay was validated both by comparing it with an ELISA and by analysing DNA samples, alkylated either 'in vitro' or 'in vivo' by dimethyl sulfate. The proposed assay proved to be sensitive, simple and reliable. It should be of value, together with other immunoassays for methylated DNA bases, in assessing human environmental exposure to alkylating agents such as nitrosamines, thereby providing a powerful tool in epidemiological investigations.

Animals↗
PubMed1990

Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine.

Oxidative damage to DNA is shown to be extensive and could be a major cause of the physiological changes associated with aging and the degenerative diseases related to aging such as cancer. The oxidized nucleoside, 8-hydroxy-2'-deoxyguanosine (oh8dG), one of the approximately 20 known oxidative DNA damage products, has been measured in DNA isolated from various organs of Fischer 344 rats of different ages. oh8dG was present in the DNA isolated from all the organs studied: liver, brain, kidney, intestine, and testes. Steady-state levels of oh8dG ranged from 8 to 73 residues per 10(6) deoxyguanosine residues or 0.2-2.0 x 10(5) residues per cell. Levels of oh8dG in DNA increased with age in liver, kidney, and intestine but remained unchanged in brain and testes. The urinary excretion of oh8dG, which presumably reflects its repair from DNA by nuclease activity, decreased with age from 481 to 165 pmol per kg of body weight per day for urine obtained from 2-month- and 25-month-old rats, respectively. 8-Hydroxyguanine, the proposed repair product of a glycosylase activity, was also assayed in the urine. We estimate approximately 9 x 10(4) oxidative hits to DNA per cell per day in the rat. The results suggest that the age-dependent accumulation of oh8dG residues observed in DNA from liver, kidney, and intestine is principally due to the slow loss of DNA nuclease activity; however, an increase in the rate of oxidative DNA damage cannot be ruled out.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1989

A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase.

1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with Ki = 11 nM. 2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme. 3. The dissociation constants (Kd) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations. 4. The Kd values for NADPH, MTX and FMTX from the complementary binary complexes (MTX.DHFR, FMTX.DHFR and NADPH.DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme. 5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10(-9) to 10(-7) M.

Animals↗
PubMed1989

Time-resolved fluoroimmunoassay of aflatoxins.

Preparation of purified and Eu-labeled antibodies specific for aflatoxins is described. Their use is illustrated by a solid-phase competitive time-resolved fluoroimmunoassay, results of which were correlated with those of an enzyme-linked immunosorbent assay based on use of the unmodified antibody to aflatoxin. This procedure is discussed as a quick, sensitive, and reliable immunoassay for use in mycotoxin screening in foodstuffs and body fluids.

Aflatoxins↗
PubMed1988

Antibodies against 7-methyldeoxyguanosine: its detection in rat peripheral blood lymphocyte DNA and potential applications to molecular epidemiology.

Polyclonal antibodies have been raised against the imidazole ring-open form of 7-methyldeoxyguanosine (7-mdGua). A combined high performance liquid chromatography/immunoassay method has been developed using these antibodies which provides a specific and sensitive way to quantitate 7-mdGua in DNA. Following enzyme hydrolysis and chromatographic purification of 7-mdGua, the adduct is quantitatively converted to the ring-open form and can be measured at levels as low as 0.05 pmol by immunoassay. With 1 mg of DNA a level below 1 adduct per 10(7) normal deoxynucleosides can be measured. Using DNA modified by radiolabeled carcinogens, a good correlation between 7-mdGua levels, as measured by immunoassay or radioactivity, was obtained. In rats treated with dimethylnitrosamine (0.4 and 1.0 mg/kg), both 7-mdGua and O6-methyldeoxyguanosine were detected in peripheral blood lymphocyte DNA. In addition the levels of both adducts at time points up to 48 h posttreatment were very similar to those seen in liver DNA from the same animals. The measurement of 7-mdGua, quantitatively the major methylation adduct, in small cell samples such as lymphocytes has great potential in determining the exposure of humans to environmental methylating agents such as nitrosamines.

Animals↗
PubMed1988

Chromosome aberrations associated with CAD gene amplification in Chinese hamster cultured cells.

Eleven sublines with increasing resistance to N-phosphonacetyl-L-aspartate (PALA) were isolated from the V79,B7 Chinese hamster cell line. Aspartate transcarbamylase activity and CAD gene copy number increased with increasing resistance of sublines. In situ hybridization with a DNA probe for the CAD gene showed that the amplified sequences resided in the terminal region of a marker chromosome with elongated q arms. This region stained homogeneously after G-banding. A high incidence of both numerical and structural chromosome aberrations was found in PALA-resistant cells. In hyperdiploid and polyploid cells, containing 2 copies of the marker chromosome, dicentrics were found at a very high frequency. As indicated by in situ hybridization and G-banding, they originated from a rearrangement involving 2 homologous marker chromosomes.

Animals↗
PubMed1988

Red blood cell sodium and potassium fluxes in psoriatic patients.

Psoriasis might be a widespread membrane disorder. Therefore, the red blood cell sodium, potassium and lithium outward fluxes (through Na-K-ATPase, Na-K-Cl co-transport, Li-Na countertransport and passive permeability), as well as the Na and K content, were studied in 31 psoriatic patients and 23 normal controls. A significant increase in intracellular potassium content, in the maximal velocity of the Na-K ATPase and of Na-K-Cl co-transport as well as in the outward passive permeability for Na were found in the psoriatic patients compared with controls. On the contrary, no differences were observed in sodium content, Li-Na countertransport and passive potassium permeability between the two groups. These results are compatible with a selective increase in inward, as well as outward, membrane permeability to sodium, which is compensated for by increased activity of the Na-K pump, and of the outward Na-K-Cl cotransport with a secondarily increased erythrocyte potassium content. They indicate that the red blood cell might be a useful model for the study of membrane transport in psoriatics.

Adult↗
PubMed1988

Detection in human cells of alkylated macromolecules attributable to exposure to nitrosamines.

Various methods for detecting DNA alkylation adducts are described briefly, with emphasis on immunoassays using antibodies against O6-methyldeoxyguanosine (O6-medGua), O4-methylthymidine (O4-meThy) and 7-methyldeoxyguanosine (7-medGua). The application of these methods to epidemiological studies is discussed, and results obtained so far on the presence of DNA alkylation adducts in human tissues are presented.

Alkylation↗
PubMedDate not supplied

A new fluorescence enhancement solution for europium-based time-resolved fluoroimmunoassays.

A cationic detergent is proposed as suitable insulating agent for the preparation of a fluorescence enhancement solution useful for europium-based time-resolved fluoroimmunoassays. Luminescence from europium ions at concentration as low as 0.5 pmol/l can be detected in solution containing 1.6 mmol/l thenoyltrifluoroacetone, 110.5 mumol/l Adogen 464 and 0.1% Tween 20, in the presence of 0.5 mol/l NaCl. A competitive TR-FIA for rabbit IgG is described, showing that the new enhancement solution allows a sensitivity comparable to that of the high performance LKB Wallac DELFIA.

Animals↗
PubMedDate not supplied

Study of 4-NQO induced mutations at the HPRT locus in CHO cell lines. Possible influences of preferential DNA-repair on the mutational spectrum.

In the attempt to determine the possible influence of excision repair processes on 4-NQO mutational spectra in mammalian cells, 4-NQO-induced mutants at the hprt locus were isolated in excision repair proficient (AA8) and deficient (UV5) CHO cell lines. DNA sequencing data on these mutants revealed that DNA repair may indeed modulate the induced mutational spectrum. In particular, more splice mutations were found in the repair proficient than in the repair deficient cells. This can be interpreted by a difference in repairability of the two principal 4-NQO G-adducts or by the existence of a transcription-coupled DNA preferential repair process.

4-Nitroquinoline-1-oxide↗