• Aristolochic acid mutagenesis: molecular clues to the aetiology of Balkan endemic nephropathy-associated urothelial cancer.

      Arlt, Volker M.; Stiborova, Marie; vom Brocke, Jochen; Simoes, Maria L.; Lord, Graham M.; Nortier, Joelle L.; Hollstein, Monica; Phillips, David H.; Schmeiser, Heinz H. (2007-11)
      Balkan endemic nephropathy (BEN) is found in certain rural areas of the Balkans and affects at least 25,000 inhabitants. Of the many hypotheses on BEN, the Aristolochia hypothesis has recently gained ground substantiated by the investigations on aristolochic acid nephropathy (AAN). On both clinical and morphological grounds, AAN is very similar to BEN. That exposure to aristolochic acid (AA) of individuals living in endemic areas through consumption of bread made with flour contaminated with seeds of Aristolochia clematitis is responsible for BEN is an old hypothesis, but one which is fully consistent with the unique epidemiologic features of BEN. Here, we propose an approach to investigate AA-induced mutagenesis in BEN that can provide molecular clues to the aetiology of its associated urothelial cancer. The molecular mechanism of AA-induced carcinogenesis demonstrates a strong association between DNA adduct formation, mutation pattern and tumour development. A clear link between urothelial tumours, p53 mutations and AA exposure should emerge as more tumour DNA from BEN patients from different endemic areas becomes available for mutation analysis. We predict that the observed p53 mutation spectrum will be dominated by AT --> TA transversion mutations as has already been demonstrated in the human p53 gene of immortalized cells after exposure to AAI and urothelial tumours from BEN patients in Croatia. Moreover, the detection of AA-specific DNA adducts in renal tissue of a number of BEN patients and individuals living in areas endemic for BEN in Croatia provides new evidence that chronic exposure to AA is a risk factor for BEN and its associated cancer.
    • Association between transcriptional activity, local chromatin structure, and the efficiencies of both subpathways of nucleotide excision repair of melphalan adducts.

      Episkopou, Hara; Kyrtopoulos, Soterios A.; Sfikakis, Petros P.; Fousteri, Maria; Dimopoulos, Meletios A.; Mullenders, Leon H. F.; Souliotis, Vassilis L. (2009-05-15)
      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.
    • DNA repair polymorphisms and the risk of stomach adenocarcinoma and severe chronic gastritis in the EPIC-EURGAST study.

      Capella, Gabriel; Pera, Guillem; Sala, Nuria; Agudo, Antonio; Rico, Francisco; Del Giudicce, Giuseppe; Plebani, Mario; Palli, Domenico; Boeing, Heiner; Bueno-de-Mesquita, H. Bas; et al. (2008-12)
      BACKGROUND: The contribution of genetic variation in DNA repair genes to gastric cancer (GC) risk remains essentially unknown. The aim of this study was to explore the relative contribution of DNA repair gene polymorphisms to GC risk and severe chronic atrophic gastritis (SCAG). Method A nested case control study within the EPIC cohort was performed including 246 gastric adenocarcinomas and 1175 matched controls. Controls with SCAG (n = 91), as defined by low pepsinogen A (PGA) levels, and controls with no SCAG (n = 1061) were also compared. Twelve polymorphisms at DNA repair genes (MSH2, MLH1, XRCC1, OGG1 and ERCC2) and TP53 gene were analysed. Antibodies against Helicobacter pylori were measured. RESULTS: No association was observed for any of these polymorphisms with stomach cancer risk. However, ERCC2 K751Q polymorphism was associated with an increased risk for non-cardial neoplasm [odds ratio (OR) = 1.78; 95% confidence interval (CI) 1.02-3.12], being ERCC2 K751Q and D312N polymorphisms associated with the diffuse type. ERCC2 D312N (OR = 2.0; 95% CI 1.09-3.65) and K751Q alleles (OR = 1.82; 95% CI 1.01-3.30) and XRCC1 R399Q (OR = 1.69; 95% CI 1.02-2.79) allele were associated with an increased risk for SCAG. CONCLUSION: Our study supports a role of ERCC2 in non-cardial GC but not in cardial cancer. A concordant result was observed for subjects with low PGA levels. XRCC1 allele was associated also with SCAG. This is the first prospective study suggesting that individual variation in DNA repair may be relevant for gastric carcinogenesis, a finding that will require further confirmation validation in larger independent studies.
    • Influence of aryl hydrocarbon- (Ah) receptor and genotoxins on DNA repair gene expression and cell survival of mouse hepatoma cells.

      Schreck, Ilona; Chudziak, Doreen; Schneider, Sandra; Seidel, Albrecht; Platt, Karl L.; Oesch, Franz; Weiss, Carsten (2009-05-17)
      The aryl hydrocarbon receptor (AhR) mediates toxicity of a variety of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. However, the underlying mechanisms and genetic programmes regulated by AhR to cause adverse effects but also to counteract poisoning are still poorly understood. Here we analysed the effects of two AhR ligands, benzo[a]pyrene (B[a]P), a DNA damaging tumour initiator and promotor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pure tumour promoter, on cell survival and on nucleotide excision repair (NER) gene expression. NER deals with so called "bulky" DNA adducts including those generated by enzymatically activated B[a]P. Therefore, the hypothesis that AhR may enhance NER gene expression to trigger DNA repair in the presence of genotoxic AhR ligands was tested. Furthermore, we investigated a potential cytoprotective effect of AhR activation by the non-genotoxic ligand TCDD against cell death induced by various genotoxins. Finally, the actions of genotoxins themselves on NER gene expression were studied. As a cell culture model we used mouse hepatoma cells (Hepa-c7) proficient for AhR and its partner protein ARNT as well as subclones deficient in AhR (Hepa-c12) or ARNT (Hepa-c4) to study involvement of AhR and ARNT in response to B[a]P and TCDD. Indeed, the mRNA levels of the two NER genes XP-C and DNA polymerase kappa were increased by B[a]P and TCDD, however, this was not accompanied by an increase in the amount of the respective proteins. Pretreatment of cells with TCDD did not reduce cytotoxicity induced by various genotoxins. Thus, in Hepa-c7 cells AhR has no major effects on the expression of these crucial NER proteins and does not prevent genotoxin-provoked cell death. As expected, the genotoxins B[a]P and cis-platin led to p53 accumulation and induction of its target p21. Interestingly, however, NER gene expression was not enhanced but rather decreased. As two NER genes, XP-C and DNA damage binding protein ddb2, are up-regulated by p53 and ultraviolet radiation in human cells these findings suggest cell type, species or lesion specific actions of p53 on DNA repair gene expression. Importantly, in cells with damaged DNA up-regulation of p53 may not suffice to enhance DNA repair gene expression.
    • The repair of melphalan-induced DNA adducts in the transcribed strand of active genes is subject to a strong polarity effect.

      Episkopou, Hara; Kyrtopoulos, Soterios A.; Sfikakis, Petros P.; Dimopoulos, Meletios A.; Souliotis, Vassilis L. (2011-09-01)
      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.