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    Keywords: CANCER ; Germany ; human ; RISK ; TIME ; ACTIVATION ; DNA ; RISK-FACTORS ; INDUCTION ; INTERVENTION ; CELL-LINES ; treatment ; SUSCEPTIBILITY ; ASSAY ; risk factors ; (-)-epigallocatechin gallate ; ADP-RIBOSE POLYMERASE ; cancer risk ; COMET ASSAY ; DAMAGE ; EPIGALLOCATECHIN GALLATE ; genotoxicity ; GREEN TEA ; GROWTH-INHIBITION ; LYMPHOCYTES ; poly(ADP-ribose) polymerase ; POLY(ADP-RIBOSYL)ATION ; POLYPHENOLS ; QUERCETIN ; repair mechanisms ; RISK FACTOR ; tea catechins
    Abstract: With regard to a future use of tea polyphenols, in intervention trials with individuals at high cancer risk, the effects of the tea ingredient (-)-epigallocatechin gallate (EGCG) on poly(ADP- ribose) (PAR) levels and on DNA damage were investigated in human lymphocytes. A dose- and time-dependent elevation of both PAR formation as assessed by quantitative immunofluorescence analysis and DNA damage as assessed by the comet assay were observed after treatment with EGCG at 20, 40 and 80 muM for 10- 240 min. Maximum levels of PAR formation and of DNA damage were observed after 10 min at all concentrations tested. Increased PAR levels were still detectable by 240 min in the 40 and 80 muM groups. At the lowest concentration, which is near the physiological peak values found after tea ingestion, PAR formation was not correlated with DNA damage. Here, EGCG led to pronounced PAR levels, whereas the comet assay was almost negative. In contrast, such marked differences in time course and extent of both genotoxicity and PAR formation following EGCG treatment were not detected after gamma-irradiation. Our results suggest that the known chemopreventive effects of EGCG, the main constituent of tea, may be partly attributed to an induction of PAR formation, (C) 2002 Elsevier Science B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 12504756
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  • 3
    Keywords: CELLS ; IN-VITRO ; IRRADIATION ; CELL ; evaluation ; Germany ; human ; GENE-EXPRESSION ; radiation ; TIME ; RESPONSES ; DNA ; CARCINOGENESIS ; INDUCTION ; KERATINOCYTES ; SKIN ; fibroblasts ; gene expression ; ASSAY ; REPAIR ; skin carcinogenesis ; genetics ; COMET ASSAY ; DAMAGE ; DNA-DAMAGE ; MAMMALIAN-CELLS ; Jun ; MORPHOLOGY ; NETHERLANDS ; ELECTROPHORESIS ; sensitivity ; heredity ; HaCaT ; PERIPHERAL-BLOOD LYMPHOCYTES ; RE ; PATTERN ; CELL-VIABILITY ; keratinocyte ; SKIN KERATINOCYTES ; INCREASE ; LEVEL ; DNA damage ; TESTS ; UV-RADIATION ; E ; STRAND BREAKS ; POLARITY ; TOXICOLOGY ; microbiology ; biotechnology ; German ; modification ; adherent HaCaT keratinocytes ; alkaline comet assay ; dose-dependence of DNA-damage induction ; HAMSTER V79 CELLS ; REPAIR CAPACITY ; trypsin effect
    Abstract: The comet assay is one of the well-accepted tests to measure radiation-induced DNA damage. The most commonly used protocols require single-cell suspensions that are embedded in agarose in order to perform electrophoresis. For adherently growing cells such as human HaCaT skin keratinocytes this method bears several problems. We show that trypsinization required for maintaining singlecell suspensions is prolonged after UV radiation and thereby reduces cell viability and allows partial repair, with the consequence of reduced damage detection after irradiation. Therefore, we here introduce a modified version of the comet assay where HaCaT cells are seeded onto comet slides 24 h before the assay and overlaid with agarose immediately after irradiation. Using this modification we are now able to reproducibly measure high DNA-damage levels (13-fold increase compared with controls) following irradiation with 60 J/cm 2 UVA as well as a dose-dependent increase of DNA damage after 10, 20 and 60 J/cm 2 UVA. Thus, by maintaining the cells in their natural configuration, i.e. adherently growing, we exclude several artefacts that are likely to influence the damage responses. These include: (i) trypsinization-dependent changes in cell morphology and polarity (clear lateral, i.e. adherent, and apical side of keratinocytes) which are likely of consequence for the gene-expression pattern, (h) trypsin- and dislodgement-induced damage reducing cell viability, and (iii) the time delay between damage induction and damage evaluation to unpredictable results due to partial repair. Since these advantages pertain to all adherently growing cells, this improved protocol is not restricted to HaCaT cells but offers great potential also with all non-haematopoietic cells for obtaining accurate results and for studying repair processes in a highly reproducible manner. (c) 2007 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17509930
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  • 4
    Keywords: validation ; QUALITY ; ASSAY ; PROMOTER ; genetics ; CHEMICAL CARCINOGENS ; statistical analysis ; ACTS ; Analysis programme ; BALB/c 3T3 cell transformation assay ; CELL-TRANSFORMATION ; William's-type protected tests
    Abstract: Validation activities of the BALB/c 3T3 cell transformation assay (CTA) - a test method used for the assessment of the carcinogenic potential of compounds - have revealed the need for statistical analysis tailored to specific features of BALB/c 3T3 CTA data. Whereas a standard statistical approach for the Syrian hamster embryo (SHE) CTA was considered sufficient, an international expert group was gathered by the European Centre for the Validation of Alternative Methods (ECVAM) to review commonly applied statistical approaches for BALB/c 3T3 CTA. As it was concluded that none of the commonly applied approaches is entirely appropriate, two novel statistical approaches were found to be recommended for the evaluation of BALB/c 3T3 CTA data accounting for possible non-monotone concentration-response relationship and variance heterogeneity: a negative binomial generalised linear model with William's-type downturn-protected trend tests and a normalisation of the data by a specific transformation allowing for application of a general linear model that estimates effects assuming a normal distribution with William's-type protected tests. Both approaches are described in this article and their performance and the quality of the results they generate is demonstrated using exemplary data. Our work confirmed that both approaches are suitable for the statistical analysis of BALB/c 3T3 CTA data and that each of them is superior to commonly used methods. Furthermore, a procedure dichotomising data into negatives and positives is proposed which allows re-testing in cases where inconclusive data are encountered. The scripts of the statistical evaluation programs written in R - a freely available statistical software - are appended including exemplary outputs (Appendix A).
    Type of Publication: Journal article published
    PubMed ID: 22178130
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  • 5
    Keywords: COLORECTAL-CANCER ; METABOLIC-ACTIVATION ; BALKAN ENDEMIC NEPHROPATHY ; urothelial cancer ; reductive activation ; CYTOCHROMES P450 1A1 ; CHINESE HERBS ; ARISTOLACTAM-DNA ADDUCTS ; ENVIRONMENTAL CARCINOGEN ; NAD(P)H/QUINONE OXIDOREDUCTASE
    Abstract: Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H:quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined. We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver. NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.
    Type of Publication: Journal article published
    PubMed ID: 24769487
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  • 6
    Keywords: CELLS ; BLOOD ; CELL ; EXPOSURE ; SITE ; SITES ; WORKERS ; MICE ; DNA ; INDUCTION ; RATS ; hepatocytes ; FREQUENCY ; FREQUENCIES ; METABOLITES ; bone marrow ; BONE-MARROW ; DNA-REPAIR ; COMET ASSAY ; LYMPHOCYTES ; DNA repair ; SINGLE-STRAND BREAKS ; INCREASE ; WEIGHT ; ADDUCT FORMATION ; STYRENE ; urinary metabolites ; LEVEL ; correlation ; ENDONUCLEASE ; 1,3-butadiene ; BUTADIENE DIOLEPOXIDE ; DNA-repair activity ; DOSE RESPONSES ; EPOXIDE HYDROLASE GENE ; micronucleus formation ; single-strand DNA breaks
    Abstract: We investigated single-strand breaks and endonuclease III-sensitive sites in DNA along with gamma-irradiation-specific DNA-repair activity in hepatocytes and frequencies of micronuclei in polychromatic bone-marrow erythrocytes of male NMRI mice (2 months old, weight 30-35 g) during sub-acute inhalation exposure to 1,3-butadiene (28 days, 500 mg/m(3)) and up to 28 days after the exposure. Concentrations of 1,3-butadiene in blood, an indicator of internal exposure, moderately increased during the exposure period. The most interesting finding was that gamma-irradiation-specific DNA-repair activity gradually increased during exposure, being significantly higher compared with control levels on days 7 and 28 of exposure (P = 0.005 and 0.035, respectively), reaching a maximum on day 1 after the termination of exposure (P = 0.003) and then returning to control levels. A significant correlation between gamma-irradiation-specific DNA-repair activity and the concentration of 1,3-butadiene in blood (R = 0.866, P = 0.050) supports a possible induction of DNA-repair activity by the exposure to 1,3-butadiene and formation of its metabolites. The initial increase in micronucleus frequency (micronuclei per 1000 cells) in the exposed mice continuously decreased from 20.4 +/- 5.1 (day 3) to 15.1 +/- 3.2 (day 28) within the exposure period, and subsequently from 12.4 +/- 5.1 to 4.6 +/- 1.6 in the period following termination of the 1,3-butadiene exposure, while micronucleus frequencies in control animals were significantly lower (from 1.7 +/- 11.5 to 4.2 +/- 0.8). (c) 2006 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16807075
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  • 7
    Keywords: CANCER ; LUNG ; LUNG-CANCER ; SYSTEM ; DNA adducts ; incidence ; SITE ; SAMPLE ; SAMPLES ; TIME ; ACTIVATION ; DNA ; DNA ADDUCT FORMATION ; ENVIRONMENTAL CONTAMINANT 3-NITROBENZANTHRONE ; BINDING ; NO ; PATTERNS ; ASSAY ; genetics ; DNA-BINDING ; METABOLIC-ACTIVATION ; NUCLEOTIDES ; POLYCYCLIC AROMATIC-HYDROCARBONS ; NETHERLANDS ; CHROMATOGRAPHY ; heredity ; LAYER ; PATTERN ; air pollution ; LEVEL ; intratracheal instillation ; CANCER INCIDENCE ; P-32-POSTLABELING ANALYSIS ; microbiology ; biotechnology ; NUCLEOTIDE ; STRAIN ; air mutagenicity ; AIR-POLLUTANT 3-NITROBENZANTHRONE ; AMBIENT AIR ; ames test ; DIESEL EXHAUST PARTICLES ; LONG-TERM EXPOSURE ; nitroarenes ; nitroaromatics ; Salmonella microsome assay ; SALMONELLA MUTAGENICITY ; TA98 ; YG1041
    Abstract: Urban particulate matter (UPM) contributes to lung cancer incidence. Here, we have studied the mutagenic activity and DNA adduct-forming ability of fractionated UPM extractable organic matter (EOM). UPM was collected with a high-volume sampler in June 2004 at two sites, one at street level adjacent to a roadway and the other inside a park within the urban area of the city of Sao Paulo, Brazil. UPM was extracted using dichloromethane, and the resulting EOM was separated by HPLC to obtain PAH, nitro-PAH, and oxy-PAH fractions which were tested for mutagenicity with the Salmonella strains TA98 and YG1041 with and without S9 metabolic activation. The PAH fraction from both sites showed negligible mutagenic activity in both strains. The highest mutagenic activity was found for the nitro-PAH fraction using YG1041 without metabolic activation; however, results were comparable for both sites. The nitro-PAH and oxy-PAH fractions were incubated with calf thymus DNA under reductive conditions appropriate for the activation of nitro aromatic compounds, then DNA adduct patterns and levels were determined with thin-layer chromatography (TLC) (32)p-postlabeling method using two enrichment procedures-nuclease PI digestion and butanol extraction. Reductively activated fractions from both sites produced diagonal radioactive zones (DRZ) of putative aromatic DNA adducts on thin layer plates with both enrichment procedures. No such DRZ were observed in control experiments using fractions from unexposed filters or from incubations without activating system. Total adduct levels produced by the nitro-PAH fractions were similar for both sites ranging from 30 to 45 adducts per 10(8) normal nucleotides. In contrast, the DNA binding of reductively activated oxy-PAH fractions was three times higher and the adduct pattern consisted of multiple discrete spots along the diagonal line on the thin layer plates. However, DNA adduct levels were not significantly different between the sampling sites. Both samples presented the same levels of mutagenic activity. The response in the Salmonella assay was typical of nitroaromatics. Although, more mutagenic activity was related to the nitro-PAH fraction in the Salmonella assay, the oxy-PAH fractions showed the highest DNA adduct levels. More studies are needed to elucidate the nature of the genotoxicants occurring in Sao Paulo atmospheric samples. (C) 2008 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18294902
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  • 8
    Keywords: BREAST-CANCER ; ACUTE LYMPHOBLASTIC-LEUKEMIA ; METHYLENETETRAHYDROFOLATE REDUCTASE ; MTHFR ; GENOTYPE ; RISK-FACTOR ; colorectal cancer risk ; METHIONINE-SYNTHASE-REDUCTASE ; COMMON MUTATION ; GENE POLYMORPHISMS ; FOLIC-ACID ; FOLATE ; Haplotype analyses ; MTRR ; ONE-CARBON METABOLISM
    Abstract: Polymorphic variants in genes involved in one-carbon metabolism, in particular of dietary folate, may modulate the risk for colorectal cancer through aberrant DNA-methylation and altered nuCIeotide synthesis and repair. In the present study, we have assessed the association of six polymorphisms and relative haplotypes in the MTHFR gene (rs1801133 and rs1801131) and in the MTRR gene (rs1801394, rs1532268, rs162036, and rs10380) with the risk for colorectal cancer in 666 patients and 1377 controls from the Czech Republic. We found that the 677 C 〉 T polymorphism in the MTHFR gene significantly decreased the risk for colorectal cancer in homozygous carriers of the variant allele (OR, 0.58:95% CI, 0.39-0.87). Also, we noted a significantly different distribution of genotypes between cases and controls for the 66A 〉 G polymorphism in the MTRR gene. In particular, homozygous carriers of the G-containing allele of this polymorphism were at an increased risk for colorectal cancer (OR, 1.39; 95% CI, 1.04-1.85). Haplotype analysis of the two MTHFR polymorphisms showed a moderate difference in the distribution of the TA haplotype between cases and controls. In comparison to the most common haplotype (CA), the TA haplotype was associated with a decreased risk for colorectal cancer (OR, 0.84; 95% CI. 0.71-0.99). No difference in the distribution between cases and controls was observed for the haplotypes based on the four polymorphisms in the MTRR gene. The present study suggests that the 677-TT genotype and the TA haplotype in the MTHFR gene may also have a role in colorectal cancer risk in the Czech population, indicating the importance of genes involved in folate metabolism with respect to cancer risk. For MTRR, additional studies on larger populations are needed to CIarify the possible role of variation in this gene in colorectal carcinogenesis.
    Type of Publication: Journal article published
    PubMed ID: 21211571
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  • 9
    Keywords: SPECTRA ; CELLS ; tumor ; CELL ; Germany ; human ; EXPOSURE ; GENE ; TUMORS ; LINES ; PATIENT ; DNA ; DOMAIN ; CARCINOGENESIS ; BINDING ; CELL-LINES ; fibroblasts ; SEQUENCE ; SEQUENCES ; treatment ; culture ; ACID ; MOUSE ; ASSAY ; MUTATION ; CELL-LINE ; LINE ; p53 ; DNA-BINDING ; MUTATIONS ; ADDUCTS ; cell lines ; P53 MUTATIONS ; CHINESE HERBS NEPHROPATHY ; DNA-ADDUCTS ; TUMOR-SUPPRESSOR ; HUMAN CANCER ; CARCINOGEN ; tumor suppressor gene ; senescence ; SIGNATURE ; SUBSTITUTION ; aristolochic acid nephropathy ; mutational spectra
    Abstract: To test hypotheses on the origins of p53 mutations in human tumors, novel strategies are needed for generating mutation spectra experimentally. To this end we developed an assay employing Hupki (Human p53 knock-in) mouse embryonic fibroblasts (HUFs). Here we examine p53 mutations induced by aristolochic acid I (AAI)), the carcinogen probably responsible for Chinese herbal nephropathy. Six immortalized cultures (cell lines) from 18 HUF primary cultures exposed at passage 1 for 48 h to 50 mu M AAI harbored p53 mutations in the human DNA binding domain sequence of the Hupki p53 tumor suppressor gene. The most frequently observed mutation was A to T transversion, corroborating our previous mutation study with AAI, and consistent with the presence of persistent AAI-adenine adducts found both in DNA of exposed patients and in DNA of AAI-exposed HUF cells. One of the mutations was identical in position (codon 139) and base change (A to T on the non-transcribed strand) to the single p53 mutation that has thus far been characterized in a urothelial tumor of a nephropathy patient with documented AAI exposure. Of the seven p53 mutations identified thus far in 〉 60 HUF cell lines that immortalized spontaneously (no carcinogen treatment), none were A:T to T:A transversions. In addition, no A to T substitutions were identified among the previously reported set of 18 mutations in HUF cell lines derived from B(a)P treatment in which transversions at G:C base pairs predominated. (c) 2006 Elsevier B.v. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16835015
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  • 10
    Keywords: CANCER ; LUNG-CANCER ; DISEASE ; RISK ; DNA ; ASSOCIATION ; POLYMORPHISMS ; BLADDER-CANCER ; MELANOMA ; MALIGNANT-MELANOMA ; XERODERMA-PIGMENTOSUM ; DNA-REPAIR GENES ; SKIN-CANCER ; BREAST-CANCER RISK ; review ; XPD POLYMORPHISMS ; BASE-EXCISION-REPAIR ; CELL REACTIVATION ASSAY ; Combined analysis ; PIGMENTOSUM GROUP-C
    Abstract: Single-nucleotide polymorphisms in different DNA-repair genes are reported to modulate risk of various cancers including melanoma. We genotyped DNA from 1186 melanoma patients and 1280 healthy controls for 13 different polymorphisms in eight DNA-repair genes. Data analyses showed that none of the polymorphisms except T241M XRCC3 was associated with an increased risk for cutaneous melanoma. Carriers of the variant alleles were associated with a decreased risk (OR 0.83; 95% CI, 0.79-0.98). Three additional polymorphisms together with T241M XRCC3 that tagged the entire gene region and the neighbouring genes KLC1, ZFYVE21 and PPP1R13B were not associated with the disease risk; neither were the inferred haplotypes. Imputation showed association of comparable magnitude with 11 non-genotyped neighbouring polymorphisms. Finally, the combination of results for all polymorphisms genotyped in the present study with published data suggests that none of the investigated polymorphisms was associated with melanoma susceptibility. We conclude that 13 non-synonymous polymorphisms in eight DNA-repair genes that are frequently investigated with respect to modulation of cancer risk in populations are not associated with susceptibility to cutaneous melanoma.
    Type of Publication: Journal article published
    PubMed ID: 20601096
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