Biomedical subjects

P Vodicka

Publications and source records attributed to P Vodicka.

At least 37 records · Page 2Linked to original sources

PubMed1999

Retinoid X receptor suppresses transformation by the v-myb oncogene.

The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-transformed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor.

Animals↗

PubMed1997

32P-postlabelling analysis of isomeric 7-alkylguanine adducts of styrene oxide.

Styrene 7,8-oxide, which reacts preferentially at the N-7 position of guanine, yielded two pairs of diastereomers 7-(1'-hydroxy-2'-phenylethyl)-dGMP (the alpha-isomer) and 7-(2-hydroxy-2-phenylethyl)-dGMP (the beta-isomer) on reaction with deoxyguanosine-3'-monophosphate (3'-dGMP). The alpha- and beta-isomers were formed in the ratio 32:68. T4 polynucleotide kinase preferentially mediated labelling of diastereomers corresponding to the beta-isomer. The beta-diastereomers showed a labelling efficiency of 52%, whereas the alpha-isomers showed a labelling efficiency of 4%. Molecular modelling experiments showed intrinsic differences between the two isomers. The torsion angles of C8-N7-2'-Ar and C8-N7-2'-1' for the alpha-isomers were 149.9 degrees and -26.4 degrees, whereas the torsion angles of C-8-N7-1'-2' and N7-1'-2'-Ar for the beta-isomers were 105.3 degrees and 179.4 degrees. The consequent interatomic distance between one of the hydrogens on the alpha-carbon and the 3'-phosphate group on the sugar residue was 5.3 A in the alpha-isomer whereas the closest distance between the hydrogens attached to the alpha-carbon and 3'-phosphate group in the beta-isomer was 6.2 A. This arrangement probably leads to steric overcrowding at the 3'-phosphate group in alpha-isomers and these are less efficiently phosphorylated than beta-isomers. In in vitro styrene oxide-modified salmon testis DNA alpha- and beta-isomers of 7-alkylguanines were formed in the ratio 37:63. The recovery of two diastereomeric beta-isomers in a 32P-postlabelling assay was 14%, but one of the diastereomers was obtained in 3-fold greater yield than the second isomer.

Animals↗

PubMed1996

The role of CYP2E1 and 2B1 in metabolic activation of benzene derivatives.

CYP2B1 and 2E1 oxidized toluene, aniline and monochlorobenzene (MCB) to water-soluble metabolites and to products covalently binding to microsomal proteins from male Wistar rats at high efficiency. Oxidation of benzene to covalently binding metabolites was catalysed by CYP2B1 and 2E1 more effectively than the formation of water-soluble metabolites, especially at low benzene levels. Thus, the formation of covalently binding products was inversely related but formation of soluble metabolites was proportional to benzene concentration. 1,4-Benzoquinone was responsible for the majority of covalent binding to microsomal proteins, being suppressed by ascorbate; 1,4-semiquinone was not important, since alpha-tocopherol did not inhibit the covalent binding and ESR showed its rapid decay, if NADPH was available. Specific antibodies and inhibitors confirmed the role of CYP2B1 and 2E1 induction. Covalent binding of benzene to DNA was largely due to benzene oxide; approximately 50% was due to N-7 guanine adduct. CYP2E1 oxidizing benzene via phenol to 1,4-hydroquinone appeared to mediate its further oxidation to 1,4-benzoquinone, which also occurred spontaneously, but was reversed in a reducing environment of microsomes with NADPH. Production of OH radicals in microsomes with NADPH was greatly stimulated by HQ and less by BQ, especially in CYP2E1 induced microsomes, although the quinones themselves failed to produce OH radicals. The quinones could act by simulation of the CYP futile cycle. Therefore, CYP2B1 and 2E1 in rats appeared essential for metabolic activation of benzene derivatives to potentially genotoxic products; BQ dominated the covalent binding of benzene to proteins, whereas DNA adducts were largely due to benzene oxide.

Animals↗

PubMed1996

7-Alkylguanine adducts of styrene oxide determined by 32P-postlabeling in DNA and human embryonal lung fibroblasts (HEL).

The modified 32P-postlabeling method was used for the detection of N-7-(2-hydroxy-phenylethyl) guanine adducts in DNA and human embryonal lung (HEL) cells treated in vitro with styrene oxide (SO). The total recovery of 7-alkylguanine adducts of styrene oxide in DNA analysed by 32P-postlabeling assay was 4.1 +/- 0.6%. The disappearance of 7-alkyldeoxyguanosine monophosphate adducts from SO-modified DNA at 37 degrees C showed a half-life of 19 h. The levels of 7-alkylguanine DNA adducts and single-strand breaks (SSBs) in DNA were determined in HEL cells treated with SO for 3 and 18 h. In the 3 h treatment there was a concentration-dependent increase of both 7-alkylguanine adducts and SSBs in DNA (r = 0.98, P = 0.012 and r = 0.99, P = 0.003, respectively). We found a significant correlation between 7-alkylguanine DNA adducts and SSBs in DNA (r = 0.98, P = 0.011). In HEL cells approximately 3-fold higher levels of both 7-alkylguanine DNA adducts and SSB in DNA were found after the SO (100 microM) treatment for 3 h than after the treatment for 18 h. A significant concentration-dependent increase was found only for SSB ( r = 0.95, P = 0.024) in the 18 h treatment with SO. There was no significant correlation between 7-alkylguanine adducts and SSBs (r = 0.72, P = 0.15). Our data suggest relatively fast removal of both 7-alkylguanine adducts of SO and DNA SSBs.

Autoradiography↗

PubMed1996

32P-postlabelling of diastereomeric 7-alkylguanine adducts of butadiene monoepoxide.

The reaction of 3,4-epoxy-1-butene (BMO) with deoxyguanosine-3'-monophosphate (3'-dGMP) resulted in the formation of two pairs of diastereomeric 7-alkyl-3'-dGMP derivatives corresponding to two isomers C¿-1 and C¿-2. The T4 polynucleotide kinase-mediated phosphorylation with [gamma-32P]-ATP showed preferential labelling of diastereo- mers of the C¿-1 isomer. The diastereomers 1 and 2 of the C¿-1 isomer had labelling efficiencies of 42%. However, the labelling efficiencies of diastereomers 3 and 4 of the C¿-2 isomer were 11 and 10%, respectively. The 32P-postlabelling of BMO-modified DNA yielded four isomers in the ratio of 4:4:1:1 with overall recoveries being 14%. The two isomers had a half-life of 270 min (C¿-1 isomer) and 300 min (C¿-2 isomer) which is in accordance with the stability predicted by other similar adduct experiments. The molecular modelling experiments showed more pronounced restricted rotation of butadiene residue in C¿-2 isomers due to steric interaction between butadiene residue at N-7 and O(6) atom of guanine than in C¿-1 isomer. The butadiene residue also leads to steric overcrowding at 3'-phosphate in C¿-2 isomer which probably restricts the access to the active site of T4 polynucleotide kinase.

Animals↗

PubMed1996

Future research directions in the use of biomarkers.

Many DNA adduct studies have been carried out in occupational groups that have been at a risk of cancer based on epidemiological results relating to exposure decades ago. Even new epidemiological publications on cancer cannot accurately address the effective exposures after about 1970. This is one justification for biomarker studies. Another justification is exposures for which epidemiological studies have not been conducted or have provided inadequate results, in spite of suspicions raised by short-term or animal experiments. The modulation of environment carcinogenesis by host polymorphism in genes for xenobiotic metabolizing and DNA repair enzymes is currently under extensive investigation. The studies relating phenotype/genotype to cancer are presently extended to various end points that may be related to cancer such as DNA adducts and cytogenetic damage. Adjustment for a metabolic phenotype or genotype may also increase the precision in the measurement. Mutations in oncogenes and tumor suppressor genes may give clues to the etiology of cancer.

Alkenes↗

PubMed1995

Styrene: from characterisation of DNA adducts to application in styrene-exposed lamination workers.

Styrene oxide, a metabolite of styrene, reacts with many centres in nucleosides but in DNA N-7-, N2- and O6-guanine are the main sites. A 32P-postlabelling method was developed for the detection of O6-styrene oxide DNA adducts from white blood cells. The method involved use of nuclease P1 and magnet transfer. The O6 adducts were detected at a fmol range with about 10% labelling efficiency. In lamination workers the O6 adducts, adjusted for adduct recovery, were detected at a level of 5 adducts/10(8) nucleotides.

DNA Adducts↗

PubMed1995

Styrene oxide-induced HPRT mutations, DNA adducts and DNA strand breaks in cultured human lymphocytes.

Styrene-7,8-oxide (SO) is the major in vivo metabolite of styrene, a widely used plastic monomer. SO has been classified as probably carcinogenic to humans. We studied the genotoxic effects of SO in human peripheral blood lymphocytes (PBL) in vitro. SO-treatment in the range of 0.05-0.6 mM for 24 h resulted in a dose-dependent decrease of cell survival and increase of HPRT mutation, O6-guanine DNA adducts and DNA strand breaks, whereas higher concentrations caused pronounced cell death. SO was a weak mutagen, inducing at most 10-20 mutants per 10(6) clonable cells (approximately 4-fold over the background) after treatment with 0.2-0.4 mM for 24 h or 6 days. The levels of DNA adducts in treated cells correlated with SO-concentrations, but only four adducts per 10(8) nucleotides were detected at the highest treatment concentrations. Yet, adducts were still detectable in cells that had been cultured for 6-8 days after treatment. SO-induced DNA strand breaks, measured with the Comet assay, were detectable after 1 h exposure to 0.05-0.1 mM. Post-treatment incubation for 24 h decreased the level of DNA strand breaks to the control level. There was no correlation between the levels of DNA adducts and frequency of HPRT mutation. The present results indicate that SO is relatively inefficient in inducing HPRT mutation and O6-guanine DNA adducts in human lymphocytes in vitro, which may be related to its pronounced cytotoxicity at concentrations above 0.4 mM. A comparison with previous in vivo data obtained by the same assays in T-lymphocytes of styrene-exposed workers suggests that chronic, low dose exposure to styrene in the work environment may be more efficient in inducing persistent DNA adducts and HPRT mutation than acute, short-term exposure.

Animals↗

PubMed1995

Biomarkers of styrene exposure in lamination workers: levels of O6-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes.

Occupational exposure to styrene was studied in nine workers of a hand lamination plant in Bohemia. Personal dosimeters were used to monitor the styrene workplace exposure, and the levels of styrene in blood and mandelic acid in urine were measured. Blood samples were taken at four occasions during a 7 month period to determine styrene-specific O6-guanine DNA adducts in lymphocytes and granulocytes, DNA strand breaks and hypoxanthine guanine phosphoribosyltransferase (HPRT) mutant frequency in T-lymphocytes. Seven administrative employees in the same factory (factory controls) and eight persons in a research laboratory (laboratory controls) were used as referents. DNA adduct levels determined by the 32P-postlabelling method in lymphocytes of laminators were remarkably constant and significantly higher (P < 0.0001) than in factory controls at all four sampling times. HPRT mutant frequencies (MF) measured by the T-cell cloning assay were higher in the laminators (17.5 x 10(-6), group mean) than in the factory controls (15.7 x 10(-6), group mean) at three of the four sampling times, but the differences were not statistically significant. However, a statistically significant (P = 0.021) difference between MF in the laminators (18.0 x 10(-6), group mean) and laboratory controls (11.8 x 10(-6), group mean) was observed at sampling time 4 (the only sampling time when this latter group was studied). This result indicates that styrene exposure may induce gene mutation in T-cells in vivo. DNA strand breaks were studied by the 'Comet assay' at the fourth sampling time. The laminators were found to have significantly higher levels of DNA strand breaks than the factory controls (P = 0.032 for tail length, TL; P = 0.007 for percentage of DNA in tail, T%; and P = 0.020 for tail moment, TM). A statistically significant correlation was also found between the levels of lymphocyte DNA adducts and all three DNA strand break parameters (TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary, no significant correlations were found between DNA adduct levels and the HPRT mutant frequencies or between the mutant frequencies and DNA strand breaks. Taken together, these results add further support to the genotoxic and possibly mutagenic effects of styrene exposure in vivo. However, no simple quantitative relationship seems to exist between the levels of styrene-induced DNA damage and frequency of HPRT mutation in T-lymphocytes.

Adult↗

PubMed1995

Uptake, distribution, and formation of hemoglobin and DNA adducts after inhalation of C2-C8 1-alkenes (olefins) in the rat.

Absorption, distribution, elimination and hemoglobin and DNA adduct formation were studied in the rat after inhalation of individual C2-C8 1-alkenes (olefins) at 300 p.p.m., 12 h a day for 3 consecutive days. The concentrations of olefins were measured in blood, lung, brain, liver, kidney and perirenal fat immediately after each exposure and 12 h after the third exposure. DNA adducts were determined by 32P-postlabeling in liver, and lymphocytes sampled immediately after the last exposure. Hemoglobin adducts were determined by GC/MS and GC/MS/MS in erythrocytes sampled immediately after the last exposure. Concentrations of 1-alkenes in blood and organs reached a steady-state level after the first 12 h exposure, and the concentrations 12 h after the last exposure were generally low, except in fat tissue. Concentrations of 1-alkenes in blood and the different tissues increased with increasing number of carbon atoms. In contrast, levels of hemoglobin and DNA adducts decreased with increasing number of carbon atoms. The decrease was most pronounced from C2 to C3. The decrease through the whole homologous series from ethene to 1-octene was most pronounced for hemoglobin adducts followed by the DNA adducts in the lymphocytes. All 1-alkenes caused formation of detectable levels of hemoglobin and DNA adducts, although the levels of hemoglobin adducts after C4-C8 exposure were low. The project illustrates important aspects of the use of biomarkers. The structure-activity approach gives possibilities for extrapolation within the homologous series.

Absorption↗

PubMed1994

Persistence of O6-guanine DNA adducts in styrene-exposed lamination workers determined by 32P-postlabelling.

A modified 32P-postlabelling method was used for the detection of styrene-specific DNA adducts in lamination workers. The persistence of O6-styrene DNA adducts was studied in DNA from lymphocytes and granulocytes of an exposed and a control group. We compared O6-adduct levels obtained from a sampling prior to vacation, after 2 weeks of vacation and after an additional 1 month of work. In granulocytes, there was no significant difference in adduct levels between the control and the exposed groups in any individual samplings. In lymphocytes of laminators the detected adduct levels were significantly higher (5.4 adducts/10(8) nucleotides) than those in the controls (1.0 adduct/10(8) nucleotides). The 2 week interruption of exposure did not influence the total O6-adduct level (4.9 adducts/10(8) nucleotides in the first sampling versus 5.1 adducts/10(8) nucleotides in the second), indicating very slow removal of the specific O6-styrene adducts from DNA.

Adult↗

PubMed1993

32P-postlabeling of DNA adducts of styrene-exposed lamination workers.

Lamination workers are exposed to high concentrations of styrene. A postlabeling method was developed for the detection of styrene-specific DNA adducts of the workers. To synthesize a standard, styrene oxide was reacted with 2'-deoxyguanosine 3'-monophosphate (dGMP) and the O6-dGMP-adduct was isolated and characterized. The human samples were assayed by the nuclease P1 version of the 32P-postlabeling technique, using magnet transfer of the adducts in chromatography. The human samples were spiked with the standards to ensure identification and quantitation. In lamination workers the O6-adducts, adjusted for adduct recovery, were detected at a level of 5 adducts/10(8) nucleotides, over five times the level in the controls.

Chromatography, Thin Layer↗

PubMed1993

Recoveries of DNA adducts of polycyclic aromatic hydrocarbons in the 32P-postlabelling assay.

The 32P-postlabelling assay for analysis of DNA adducts of chemical carcinogens has been applied in a large number of experimental animal and human studies. Most human studies have dealt with occupational and environmental exposures to polycyclic aromatic hydrocarbons (PAHs). The postlabelling assay does not allow direct chemical identification, and most studies with this method have not been performed in a quantitative way. Very little is therefore known about the identity and absolute levels of adducts, which are important contributors to the process of risk identification and quantitation. In the present study it was, therefore, decided to test some parameters suspected to affect recoveries of adducts in the phosphorylation step of the assay. For this purpose 12 different PAHs were reacted individually and in a mixture with DNA in the presence of a rat liver S9 metabolizing system. Different concentrations of ATP, calcium chloride and polynucleotide kinase were tested using the nuclease P1 enhancement. We found that each factor contributed to adduct recovery and that optimal conditions could be defined. Diluting the modified DNA samples up to 1000 times had little influence on the recoveries of adducts. Comparing the nuclease P1 and the butanol extraction procedures for adduct purification showed that both methods gave similar patterns and levels of major adducts. The absolute recoveries in postlabelling, based on 3H-binding of radiolabelled compounds, were for most of the tested compounds relatively low. The fact that the nuclease P1 and the butanol extraction procedures gave similar recoveries points towards common factor(s) involved in the reduction of the recovered adduct levels. Based on the observed recoveries the conclusion can be drawn that when postlabelling related adducts in human samples the true total adduct levels can be considerably underestimated, even if optimal conditions are used.

Animals↗

PubMed1993

Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts.

A further development of an HPLC method to analyze 32P-postlabeled DNA adducts is presented. The method is based on on-line detection of 32P radioactivity after separation by reversed-phase chromatography. The method has an advantage in that the postlabeling mixture can be injected directly into the HPLC system without any prior purification, with the background radioactivity on a low level. The analysis includes the whole range of substances from orthophosphate to non-polar DNA adducts, which makes it possible to analyze normal nucleotides and ATP together with DNA adducts. The analytical system has a high reproducibility and separates complex mixtures of DNA adducts. The slightly lower sensitivity compared to the TLC method is compensated for by the possibility of injecting large amounts of DNA into the system without affecting the analytical properties. The system can be applied to different DNA adducts as well as complex mixtures of DNA adducts.

2-Acetylaminofluorene↗

PubMed1993

32P-postlabelling of DNA adducts in styrene oxide-modified DNA and in workers exposed to styrene.

Lamination workers are exposed to large amounts of styrene. A postlabelling method was developed in order to detect DNA adducts in white blood cells from such workers. First, styrene oxide was reacted with DNA, and the adducts were characterized by the nuclease P1 version of the 32P-postlabelling technique. The adducts in human samples were transferred magnetically during chromatography. The 2'-deoxyguanosine 3'-monophosphate (dGMP) adduct standards, including N2 and O6 adducts, were shown to be resistant to the action of nuclease P1. The O6 adducts were detected in the femtomole range at about 10% labelling efficiency. In lamination workers, the level of O6 adducts, adjusted for adduct recovery, was 5/10(8) nucleotides, which was five times the level in controls.

Autoradiography↗

PubMed1993

Testing of quantitative parameters in the 32P-postlabelling method.

The 32P-postlabelling technique involves many steps that need to be carefully controlled in order to obtain a reliable quantitative determination of DNA adducts. We have studied several of the parameters involved in the DNA digestion procedures as well as those concerned in the phosphorylation reaction. Since adducts behave in very different ways in the labelling reaction, an individual protocol has to be worked out for each particular type of adduct. Quantitation is usually possible only if a synthesized standard of the adduct under investigation is run in parallel to the DNA samples throughout the whole procedure.

Adenosine Triphosphate↗

PubMed1992

Determination of malonaldehyde-modified 2'-deoxyguanosine-3'-monophosphate and DNA by 32P-postlabelling.

The 32P-postlabelling assay was used to determine adducts arising upon the reaction of malonaldehyde with 2'-deoxyguanosine-3'-monophosphate. The adducts formed were isolated, structurally characterized and identified as 3-(2-deoxy-beta-D-erythro-pentafuranosyl)pyrimido[1,2-alpha] purin-10(3H)-one. The kinetics of phosphorylation by T4 polynucleotide kinase was studied using 500 fmol of the synthesized standard and found to reach its maximum after 1 h of incubation. A 60% labelling efficiency was obtained at low concentrations of substrate. The adducted substrate was detected at the sub-femtomolar level. Sensitivity of the adducts towards nuclease P1 3'-dephosphorylation was also tested. The same adduct could be detected from calf thymus DNA that had been reacted in vitro with malonaldehyde, and in DNA isolated from mice treated with [14C]malonaldehyde. DNA adducts formed in vitro were isolated after enzymatic digestion to mononucleotides followed by HPLC fractionation or nuclease P1 digestion of normal nucleotides. A combination of the two procedures proved to be the method of choice for the isolation of the malonaldehyde-DNA adducts formed in vivo prior to applying the 32P-postlabelling assay.

Animals↗

PubMed1991

The prospects of the development of the method for monitoring of occupational exposure to some alkylating agents.

DNA-adduct formation, depurination and imidazole ringopening were followed in vitro using styrene oxide, ethyleneimine and dimethyl sulfate. Depurination was found to be about 50 times faster in nucleosides than in double-stranded DNA. The half-lives of depurination in DNA were 3 times faster for 7-(2-aminoethyl)guanine as compared to 7-methyl- and 7-(2-hydroxy-2-phenylethyl)deoxyguanosine. In neutrally 7-methylguanine was released some 60 times faster than that of guanine and adenine. This apparent discrepancy in depurination between alkylated and intact bases suggests the possibility of developing a sensitive method for monitoring of DNA alkylations formed by electrophillic chemicals, which might be based on labelling of apurinic sites and utilized for in vivo studies as well.

Alkylating Agents↗