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ABSTRACT: We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 μg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.
Mutagenesis 05/2012; 27(5):589-97. · 3.18 Impact Factor
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ABSTRACT: DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable. Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O(6)-methylguanine-DNA-methyltransferase (MGMT), which repairs the O(6)-alkylguanine-type of adducts induced in DNA by alkylating genotoxins; and (2) apurinic/apyrimidinic endonuclease 1 (APE 1), which participates in base excision repair (BER) by causing a rate-limiting DNA strand cleavage 5' to the abasic sites. The MGMT assay makes use of the fact that: (a) the enzyme works by irreversibly transferring the alkyl group from the O(6) position of guanine to a cystein residue in its active site and thereby becomes inactivated and (b) that the free base O(6)-benzylguanine (BG) is a very good substrate for MGMT. In the new assay, cell extracts are incubated with BG tagged with biotin and the resulting MGMT-BG-biotin complex is immobilized on anti-MGMT-coated microtiter plates, followed by quantitation using streptavidin-conjugated alkaline phosphatase and a chemiluminescence-producing substrate. A one-step/one-tube phenotypic assay for APE1 activity has been developed based on the use of a fluorescent molecular beacon (partially self-complementary oligonucleotide with a hairpin-loop structure carrying a fluorophore and a quencher at each end). It also contains a single tetrahydrofuran residue (THF) which is recognized and cleaved by APE1, and the subsequently formed single-stranded oligomer becomes a fluorescence signal emitter. Both assays are highly sensitive, require very small amounts of protein extracts, are relatively inexpensive and can be easily automated. They have been extensively validated and are being used in the context of large-scale molecular epidemiology studies.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 05/2012; 736(1-2):25-32. · 2.85 Impact Factor
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Dimitra T Stefanou,
Hara Episkopou, Soterios A Kyrtopoulos,
Aristotelis Bamias,
Maria Gkotzamanidou,
Christina Bamia,
Christina Liakou,
Margarita Bekyrou,
Petros P Sfikakis,
Meletios-Athanasios Dimopoulos,
Vassilis L Souliotis
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ABSTRACT: WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Previous studies have indicated that the levels of DNA damage induced in peripheral blood mononuclear cells by the alkylating drugs melphalan, cisplatin and carboplatin can serve as useful biomarkers predictive of the therapeutic response of cancer patients to these drugs. WHAT THIS STUDY ADDS • In the present study we developed a quantitative PCR-based assay, for the measurement of DNA damage. The advantages of this methodology are based on: ➢ its far greater sensitivity (about 250 times) than the traditional Southern blot-based method (the detection limit is ∼10-20 lesions/10(6) nucleotides from the equivalent DNA of ∼8000 cells), ➢ its simplicity and speed (results obtained within ∼8h), ➢ its excellent reproducibility, with a coefficient of variance of 10-15% for different DNA preparations from similarly treated cells, ➢ its requirement for only minute amounts of material, and ➢ the avoidance of radioisotope labeling. • Moreover, emphasis was given to translate basic research findings into clinical practice through the validation of this assay for prediction of clinical outcome in multiple myeloma patients. AIM In order to develop and validate a simple, sensitive and rapid method for the quantitation of alkylating drug-induced DNA damage. METHODS HepG2 cells and blood samples were treated with alkylating drugs (melphalan, cisplatin, carboplatin). Gene-specific damage was examined using Southern blot and a multiplex long quantitative PCR (QPCR) carried out in a 7 kb fragment (part of the p53 gene) and a 0.5 kb fragment (part of the IFN-β1 sequence; internal standard). RESULTS The extent of PCR amplification of a p53 fragment was inversely proportional to the treatment concentrations of all anticancer drugs examined, indicating a dose-related inhibition by the DNA adducts formed. Parallel analysis of the same samples using both Southern blot and QPCR showed that the DNA adducts measured by QPCR corresponded to the interstrand cross-links in the case of melphalan, and to total drug-induced lesions in the case of the platinum drugs. The detection limit was ∼10-20 lesions/10(6) nucleotides using DNA from ∼8000 cells. The method is about 250 times more sensitive than the Southern blot-based method and the reproducibility is excellent, with an intraday coefficient of variance (CV) of 5-9% and an interday CV of 4-12%. Application of the QPCR assay to ex vivo melphalan-treated peripheral blood mononuclear cells from multiple myeloma patients, showed that the positive predictive value of this assay for clinical response to melphalan therapy was 92.9%. CONCLUSION The PCR-based assay developed in this study can be used for the selection of cancer patients more likely to benefit from therapeutic treatment with alkylating drugs.
British Journal of Clinical Pharmacology 03/2012; 74(5):842-53. · 2.96 Impact Factor
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ABSTRACT: To investigate the mechanisms of the therapeutic action and drug resistance to the nitrogen mustard melphalan, melphalan-induced DNA damage repair and chromatin structure were examined along the p53, N-ras and d-globin gene loci in cells carrying different repair activities. In nucleotide excision repair-deficient XP-A cells, similar levels of adducts were found in all fragments examined, indicating uniform distribution of DNA damage. In both, repair-proficient CS-B and XP-C cells, faster repair was observed in regions inside the transcribed N-ras and p53 genes, compared to regions on both sides outside of the genes, while no such difference was observed for the inactive d-globin gene. Moreover, very fast adduct repair on the transcribed strand of the active genes was seen immediately downstream of the transcription start site, together with a steeply decreasing gradient of repair efficiency along the gene towards the 3'-end. In all cells analyzed, the above variation in DNA repair efficiency was paralleled exactly by the variation in the degree of local chromatin condensation, more relaxed chromatin being associated with faster repair. Similar results were obtained using peripheral blood mononuclear cells from healthy volunteers, suggesting that the existence of a repair gradient along transcribed genes may be a universal phenomenon. In conclusion, these findings demonstrate that the repair of melphalan adducts in the transcribed strand of active genes is subject to a strong polarity effect arising from variations in the chromatin structure.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 09/2011; 714(1-2):78-87. · 2.85 Impact Factor
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ABSTRACT: Investigations of the presence of the precarcinogenic DNA adduct O⁶-methylguanine (O6-meG) in humans and its association with exposure or cancer risk have been hindered by the absence of analytic methods of adequate sensitivity and throughput. We report the development, validation, and application of an ELISA-type assay for O6-meG appropriate for large-scale population studies.
In the new analytic method, restriction enzymes are used to digest DNA to fragments of size expected to contain no more than one O6-meG residue. Anti-adduct antisera are used to transfer O6-meG-containing fragments to a solid surface, where they are detected using anti-ssDNA antisera, the high ratio of normal nucleotides to adducts providing a strong signal enhancement.
An assay with a limit of detection of 1.5 adducts/10⁹ nucleotides using 10 μg of DNA, a dynamic range of approximately two orders of magnitude and satisfactory precision and accuracy characteristics was established and validated. Analysis of samples from 120 subjects from the Rhea mother-child cohort in Crete led to the detection of O6-meG in 70% of maternal and 50% of cord blood buffy coat samples at mean levels of 0.65 and 0.38 adducts/10⁸ nucleotides, respectively.
The frequent observation of O6-meG in human DNA is compatible with dietary compounds (e.g. N-nitroso compounds or their precursors), or endogenous processes being responsible for the formation of this adduct.
The new assay opens the way for large-scale population studies of O6-meG as a biomarker of exposure or risk. The approach used in this assay can, in principle, be extended to any DNA adduct for which suitable antisera are available.
Cancer Epidemiology Biomarkers & Prevention 11/2010; 20(1):82-90. · 4.12 Impact Factor
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ABSTRACT: The repair of melphalan-induced N-alkylpurine monoadducts and interstrand cross-links was examined in different repair backgrounds, focusing on four genes (beta-actin, p53, N-ras, and delta-globin) with dissimilar transcription activities. Adducts were found to be substrates for both global genome repair (GGR) and transcription-coupled repair (TCR), with TCR being less efficient than GGR. In nucleotide excision repair-deficient cells, adducts accumulated to similar levels in all four genes. The repair efficiency in different gene loci varied in a qualitatively and quantitatively similar way in both GGR-deficient and TCR-deficient backgrounds and correlated with transcriptional activity and local chromatin condensation. No strand-specific repair was found in GGR(+)/TCR(+) cells, implying that GGR dominated. Adducts were lost over two sharply demarcated phases: a rapid phase resulting in the removal within 1 hour of up to approximately 80% of the adducts, and a subsequent phase with t(1/2) approximately 36 to 48 hours. Following pretreatment of cells with alpha-amanitin, the rate of transcription, the state of chromatin condensation, and the repair efficiencies (both TCR and GGR) of the transcribed beta-actin, p53, and N-ras genes became similar to those of the nontranscribed delta-globin gene. In conclusion, a continuous, parallel variation of the state of transcription and local chromatin condensation, on one hand, and the rates of both GGR and TCR, on the other hand, have been shown.
Cancer Research 06/2009; 69(10):4424-33. · 7.86 Impact Factor
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ABSTRACT: Olive oil, one of the oldest vegetable oils consumed without any refining, is associated with a reduced risk of a number of common cancers. Minor constituents of virgin olive oil have been suggested to be among the major chemopreventive components. A brief overview is presented of recent findings concerning the bioavailability of certain important olive oil minor components including efficient antioxidant polyphenols, the triterpene hydrocarbon squalene and beta-sitosterol, considered as putative nutritional biomarkers, in relation to the incidence of cancer.
European Journal of Nutrition 06/2008; 47 Suppl 2:69-72. · 2.75 Impact Factor
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ABSTRACT: As new therapeutic options for multiple myeloma (MM) emerge, identification of biological markers which could predict clinical response to standard treatment with high-dose melphalan (HDM) supported by autologous stem cell transplantation (ASCT) becomes more important.
Melphalan-induced damage formation and repair of monoadducts and interstrand cross-links in the p53 gene were studied in peripheral blood mononuclear cells obtained from 32 patients prior to therapy. The same studies were performed in the peripheral blood cells of these patients immediately after subsequent HDM administration. Clinical response and time to progression were correlated with molecular endpoints obtained in vitro.
Values for all molecular end-points examined in vitro were highly correlated with the respective in vivo results within individual patients. All in vitro end-points indicative of increased DNA damage and slower repair capacity were predictive of a favorable response to HDM; the area under the curve of total adducts (AUC-TA) had the highest predictive ability. Using the cut-off value of 736 adducts/10(6) nucleotides x h for the AUC-TA, the positive predictive value for clinical response to HDM was 100%. Moreover, patients with an AUC-TA equal to or higher than this cut-off value had significantly longer times to progression than had patients with an AUC-TA lower than the cut-off value (hazard ratio 0.19; 95% confidence intervals 0.06 to 0.60).
An in vitro assay to quantify melphalan-induced p53-specific damage formation/repair can be used to select those patients with MM who are more likely to benefit from HDM supported by ASCT.
Haematologica 12/2007; 92(11):1505-12. · 6.42 Impact Factor
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ABSTRACT: To investigate the molecular mechanisms of action of the nitrogen mustard melphalan in patients treated for multiple myeloma, the in vivo induction and repair of melphalan-induced DNA damage was measured in genes with different transcriptional activity (b-actin>p53>N-ras>d-globin) from leukocytes of 20 multiple myeloma patients following chemotherapeutic administration of high-dose melphalan (200mg/m(2)) and autologous blood stem cell transplantation. Heterogeneous repair was found among the studied genes. The extent of repair was always in the order: b-actin>p53>N-ras>d-globin, correlating with the gene transcriptional state. Similar findings were obtained using peripheral blood mononuclear cells (PBMC) from healthy volunteers following in vitro treatment with melphalan, indicating that these results are not malignant disease-specific. Following in vitro treatment of PBMC from healthy volunteers with alpha-amanitin, an inhibitor of RNA polymerase II that can also induce condensation of chromatin structure, a significant inhibition of the removal of melphalan-induced damage in the three active genes but not in the silent d-globin gene was found, suggesting that transcription and/or chromatin structure may play important roles in the preferential DNA repair. When the in vivo DNA damage formation and repair in multiple myeloma patients following chemotherapeutic administration of melphalan was measured in the two strands of the active genes, no strand bias was found, indicating that the global genome repair subpathway of nucleotide excision repair may play a crucial role in the repair of these adducts. These results were also confirmed in PBMC from healthy volunteers following in vitro treatment with melphalan. Using micrococcal nuclease digestion of nuclei isolated from PBMC of multiple myeloma patients before the chemotherapeutic treatment, as well as from PBMC of healthy volunteers, we probed the chromatin structure in each gene and found that the "looseness" of the chromatin structure correlated with the levels of the gene-specific repair, being again in the order: b-actin>p53>N-ras>d-globin. To conclude, the in vivo gene-specific repair of melphalan-induced damage in humans is greatly affected by the local chromatin structure.
DNA Repair 08/2006; 5(8):972-85. · 4.14 Impact Factor
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ABSTRACT: The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.
Chemical Research in Toxicology 06/2006; 19(6):868-78. · 3.78 Impact Factor
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Soterios A Kyrtopoulos
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ABSTRACT: During the past 30 years biomarker-based approaches have been employed in the area of environmental carcinogenesis research with the expectation of refining exposure assessment, providing tools for the early detection of disease-related changes and their association with environmental and genetic factors and, thus, facilitating improved understanding of the etiology of human cancer. Great advances have been achieved in the development of analytical methodologies for the measurement of various types of biomarkers, including chemical-specific biomarkers of exposure, as well as in the characterisation of genetic variation in many genes of relevance to carcinogenesis and the assessment of its impact on cancer risk. However, despite the enormous effort invested in these investigations, the progress achieved in terms of the identification of environmental or genetic factors which affect substantially cancer risk among the general population is relatively limited. It is suggested that progress towards this goal may be facilitated by a concerted effort to promote thorough validation of chemical-specific biomarkers of exposure for which adequate analytical methodologies are available, the development of reliable and high-throughput phenotypic assays of biomarkers of susceptibility and the formulation of a systems biology approach to the analysis of the modulation of biomarkers by environmental and genetic factors.
Toxicology Letters 04/2006; 162(1):3-15. · 3.23 Impact Factor
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ABSTRACT: The protein O6-alkylguanine-DNA alkyltransferase (Atase) is responsible for the repair of DNA lesions generated by several clinically important anti-cancer drugs; this is manifest as active resistance in those cancer cell lines proficient in Atase expression. Novel O6-substituted guanine analogues have been synthesized, bearing acidic, basic and hydrogen bonding functional groups. In contrast to existing O6-modified purine analogues, such as methyl or benzyl, the new compounds were found to resist repair by Atase even when tested at concentrations much higher than O6-benzylguanine, a well-established Atase substrate active both in vitro and in vivo. The inactivity of the new purines as covalent substrates for Atase indicates that agents to deliver these groups to DNA would represent a new class of DNA-modifying drug that circumvents Atase-mediated resistance.
European Journal of Medicinal Chemistry 04/2006; 41(3):330-9. · 3.35 Impact Factor
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Panagiotis Georgiadis,
Jan Topinka,
Dimitris Vlachodimitropoulos,
Melpomeni Stoikidou,
Maria Gioka,
Georgia Stephanou,
Herman Autrup,
Nikolaos A Demopoulos,
Klea Katsouyanni,
Radim Sram, Soterios A Kyrtopoulos
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ABSTRACT: CYP1A1 plays an important role in the metabolic activation of polycyclic aromatic hydrocarbons (PAH), carcinogenic components of air pollution. The influence of CYP1A1 genotype (*2A, *2B and *4) on the levels of lymphocyte bulky DNA adducts and the frequency of cells with aberrant chromosomes was assessed in 194 non-smoking subjects in whom recent exposure to environmental tobacco smoke (ETS) and airborne particulate-associated PAH were measured during two consecutive seasons (winter and summer). While CYP1A1*4 had no consistent effect on either biomarker of genetic damage, the levels of both biomarkers responded in a parallel fashion to changes in exposure/CYP1A1*2A genotype combinations during both seasons. Specifically, the levels of both biomarkers were increased in carriers of at least one CYP1A1*2A allele, as compared with CYP1A1*1 homozygotes, in subjects with ETS exposures >0.8 h/day during the previous 4 days and mean personal exposure to benzo[a]pyrene <0.9 ng/m3 during the previous 24 h (all P < 0.05). Outside these exposure limits the differential effect in CYP1A1*2A variants was lost. Although the numbers of subjects with the CYP1A1*2B polymorphism was small, the same trend appeared to be followed in this case. These effects are interpreted as resulting from differential induction of CYP1A1 expression in CYP1A1*2A and CYP1A1*2A/*2B carriers by components of ETS-polluted air at levels of exposure readily suffered by large segments of the general population and suggest that subjects with these genotypes may have increased susceptibility to the genotoxic effects of ETS.
Carcinogenesis 02/2005; 26(1):93-101. · 5.70 Impact Factor
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ABSTRACT: Following administration to rats of various doses of N-nitrosodimethylamine (NDMA), O(6)-methylguanine (O(6)-meG) was lost from the DNA of four tissues (liver, white blood cells, lymph nodes, bone marrow) over two, sharply demarcated phases with substantially differing repair rates. Repair during each phase followed approximately first-order kinetics in O(6)-meG, even after a high dose of NDMA which caused substantial depletion of O(6)-alkylguanine-DNA alkyltransferase (AGT), a suicide repair protein. This is compatible with rate-determining adduct repair being brought about by a distinct, minor pool of AGT molecules which is rapidly replenished by de novo AGT synthesis. Similar biphasic repair kinetics were also observed in HepG2 cells treated in vitro with NDMA. In this case, the first phase of repair was inhibited by alpha-amanitin, an inhibitor of RNA polymerase II-mediated transcription. However, no dependence on transcriptional activity was found when O(6)-meG repair in specific gene sequences with different transcriptional status in rat liver was examined, suggesting that the effects of alpha-amanitin in HepG2 cells did not reflect inhibition of preferential repair of transcribed sequences. Repair was also examined in rat liver hepatocytes and non-parenchymal cells separately after administration of NDMA at non-AGT depleting doses. Within each cell-population, the repair followed single phase, first-order kinetics, with adduct loss from AGT-rich hepatocytes being significantly faster than from the relatively AGT-deficient non-parenchymal cells. In conclusion, differences in the AGT content of different cell subpopulations in the liver (and probably in other tissues), as well as additional cellular factors affecting repair efficiency, appear to determine the observed variation in the kinetics of repair of O(6)-meG. The additional cellular factors involved appear not to be related to the transcriptional state of the sequences being repaired, but may reflect different states of chromatin condensation.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2005; 568(2):155-70. · 2.85 Impact Factor
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ABSTRACT: Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/2002; 500(1-2):75-87. · 2.85 Impact Factor
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ABSTRACT: N-nitrosodimethylamine (NDMA) is a human cancer initiator suspect. Ethanol, a cancer risk factor, may synergize with nitrosamines by suppressing hepatic clearance, to increase internal exposure. A limitation to these hypotheses is lack of activation of NDMA by many rodent tissues. However, systematic primate studies are lacking. Patas monkeys were utilized to investigate NDMA activation by primate tissues in vivo, generating the promutagenic DNA lesion O6-methylguanine (O6-meG). Adult monkeys received 0.1 mg/kg NDMA by gavage, in some cases preceded by ethanol. Four hours after NDMA only, O6-meG was detected in DNA from all tissues. Levels were highest in gastric mucosa and liver and were only about 50% lower in DNA from white blood cells, esophagus, ovary, pancreas, urinary bladder and uterus. With ethanol co-exposure, amounts of O6-meG increased at least 2-fold in all tissues except liver. The largest effect was in esophagus (17-fold increase), followed by ovary, large intestine, urinary bladder, spleen and cerebellum (9- to 13-fold increases), and uterus, cerebrum and brain stem (7- to 8-fold increases). Alkylguanine alkyltransferase activities varied over a 30-fold range and were highest in liver and stomach. Thus primate tissues, especially those of the gastrointestinal and urogenital organs, are sensitive targets for DNA adduct damage due to NDMA, and ethanol co-exposure leads to striking increases in adducts. Our data support epidemiology implicating nitrosamines in causation of cancers of stomach and other organs, and alcohol as enhancing internal exposure to nitrosamines. © 1996 Wiley-Liss, Inc.
International Journal of Cancer 03/1996; 66(1):130 - 134. · 5.44 Impact Factor
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ABSTRACT: ECNIS is a Network of Excellence within the European Union’s Sixth Framework Programme, Priority 5: Food Quality and Safety. It brings together some of the best European research groups in a concerted effort to achieve improved understanding of the environmental causes of cancer, of the potential of diet to prevent cancer, and of the ways by which heredity can affect individual susceptibility to carcinogens, with the ultimate aim of reducing the cancer burden in Europe. ECNIS is coordinated by Prof. Konrad Rydzynski, The Nofer Institute of Occupational Medicine, Sw. Teresy 8, 91-348 ¸Lodz, Poland. This review has been prepared as part of ECNIS Work Package 10: Mechanistic research to support cancer hazard and risk assessment.
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Margaret M. Manson,
Jakob Linseisen,
Sabine Rohrmann,
Theodore G. Sotiroudis, Soterios A Kyrtopoulos,
John D Hayes,
Michael O Kelleher,
Ian M Eggleston,
Theo M. de Kok,
Simone G. van Breda,
Joseph Rafter
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ABSTRACT: ECNIS is a Network of Excellence within the European Union’s Sixth Framework Programme, Priority 5: Food Quality and Safety. It brings together some of the best European research groups in a concerted effort to achieve improved understanding of the environmental causes of cancer, of the potential of diet to prevent cancer and of the ways in which heredity can affect individual susceptibility to carcinogens, with the ultimate aim of reducing the cancer burden in Europe. ECNIS is coordinated by Prof. Konrad Rydzyƒski, Nofer Institute of Occupational Medicine,ul. Sw. Teresy 8, 91-348 Lodz, Poland. This review has been prepared as part of ECNIS Work Package 9: Mechanisms of modulation of cancer by dietary factors.
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ABSTRACT: 4.1. Biomarkers for intake of heterocyclic aromatic amines 4.2. Polycyclic aromatic hydrocarbons in food 4.3. Biomarkers of N-nitroso compounds 4.4. Acrylamide 4.5. Alcohol biomarkers 4.6. Aflatoxins and other mycotoxins The mutagenic and carcinogenic heterocyclic aromatic amines are formed from precur-sors in meat and fish at temperatures exceeding 130°C and bind covalently to DNA after metabolic activation. Heterocyclic aromatic amines in urine have short half-lives but could be used to validate intake as estimated by questionnaires. Blood protein adducts and DNA adducts from various tissues have also been analysed. No epidemiological studies have yet been conducted in humans that have examined the association between heterocyclic aromatic amine exposure assessed by means of any biomarker and the risk of cancer. Polycyclic aromatic hydrocarbons, formed by the incomplete combustion of organic matter, are ubiquitous contaminants of the environment. There have been two main approaches to measurement of polycyclic aromatic hydrocarbons in complex matrices such as food: determination of around 15–20 polycyclic aromatic hydrocarbons, including carcinogenic compounds, or measurement of benzo[a]pyrene as a surrogate for all polycyclic aromatic hydrocarbons. The first approach gives a truer picture of the overall burden of these compounds in food; however, benzo[a]pyrene, because of its carcinogenic potency in experimental animals, represents a biologically significant measure. Most types of food contain measurable levels of polycyclic aromatic hydrocarbons and dietary exposure can be a significant effect in studies designed to determine occupational exposure or exposure due to urban pollution. N-nitroso compounds, and especially alkyl nitrosamines, are well known experimental carcinogens. Nitrosamines are present in significant quantities in tobacco smoke, while dimethylnitrosamine is also found in nitrate- or nitrite-treated foods. N-nitroso com-pounds can be formed endogenously at significant levels. Most epidemiological studies attempting to associate exposure to N-nitroso compounds and various human cancers have been inconclusive. The main problem was the inadequacy of methods for estimation not only of external exposure but, more importantly, of endo-genous exposure to N-nitroso compounds. Large-scale molecular epidemiological studies to determine the carcinogenic risk associated with the widespread presence in human DNA of O6-methylguanine, which plays an important role in mutagenesis, carcinogenesis and cytotoxicity by methylating agents, are lacking due to the lack of high throughput, high sensitivity assays for this adduct. The quantitatively most important DNA alkylation lesion N-7-methylguanine is not directly premutagenic, but can undergo spontaneous depurination to form mutagenic apurinic sites. N-7-Methylguanine accumulates more in tissues from smokers than nonsmokers, indicating that this biomarker could be used as an internal dosimeter for exposure to nitrosamines. Extensive research on tobacco-specific nitrosamines has failed to provide conclusive evidence of their role in human cancer, despite their being potent rodent carcinogens. The urinary 4-nitrosomethylamino)-1-(3-pyridyl)-1-butanone metabolites 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanol and its glucuronide are absolutely specific for tobacco exposure. Significant amounts of acrylamide can occur in certain food items high in carbohydrates and amino acids after heating. Acrylamide has been classified by IARC as “probably carcinogenic to humans” and the EU has classified it as a “Category 2 carcinogen and cate-gory 2 mutagen”. The adduct of acrylamide itself to the N-terminal valine in haemoglobin and to some extent the corresponding adduct of glycidamide have been applied in human studies to assess exposure. Epidemiological investigations have not shown an increased risk from dietary exposure but larger studies of populations with more varied diets are needed; in addition data from intervention studies and on urinary metabolites and cytogenetic effects would be useful Biomarkers of alcohol may potentially be used to address the limitations of questionnaires and interviews in exposure assessment in epidemiological studies of alcohol as a risk factor for cancer. The available biomarkers differ in sensitivity, specificity, ease of assay, and the time period that they reflect and none alone is ideal; combinations of various markers may allow for finer assessment of alcohol exposures in the future. Mycotoxins are ubiquitous toxic secondary metabolites of a number of species of moulds, and occur in foods and animal feeds. Naturally occurring aflatoxins are a cause of hepato-cellular carcinoma. Most European countries have imposed limits for aflatoxin B1 in foods and for aflatoxin M1 in milk. Urinary aflatoxin B1-N-7-guanine is an excellent biomarker for studies of acute exposure but does not reflect chronic intake of aflatoxin. Aflatoxin B1-albumin adducts are currently the most widely used biomarkers of aflatoxin in epidemiological studies. Assessment of functional polymorphisms in CYP3A4 and in other enzymes involved in the activation and detoxification of aflatoxin B1 may be used as markers of susceptibility to aflatoxins. The codon 249 mutation in p53 must be used cautiously as a marker of exposure to aflatoxin until evidence has been obtained from studies measuring both aflatoxin B1 adducts and mutations in the same individuals. ECNIS Network of Excellence
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ABSTRACT: Single residues of O6-methylguanine (O6-meG) were introduced into the first or second position of codon 12 (GGC; positions 12G1 or 12G2, respectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors. After transformation of E.coli , higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT. Taken together, these observations suggest reduced AGT repair at 12G2. Kinetic analysis of in vitro DNA replication in the same sequences using E.coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis. By constructing vectors which direct methyl-directed mismatch repair to the (+) or the (−) strand and comparing the MPF values in bacteria proficient or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O6-meG from O6-meG:C pairs, this repair mechanism can affect O6-meG mutagenesis by repairing G:T pairs generated through AGT-induced demethylation of O6-meG:T replication intermediates.
