DNA double-strand break repair genotype and phenotype and breast cancer risk within sisters from the New York site of the Breast Cancer Family Registry (BCFR).

PURPOSE: We previously observed that poor DNA repair phenotype is associated with increased breast cancer (BC) risk within families. Here, we examined whether genetic variation in double-strand break repair (DSBR) genes is associated with BC risk and if genotypes are related to phenotype in unaffected women. METHODS: Using data from the New York site of the Breast Cancer Family Registry, we investigated 25 single-nucleotide polymorphism (SNPs) involved in DSBR using biospecimens from 337 BC cases and 410 unaffected sister controls. RESULTS: Genotypes in XRCC4 were associated with BC risk, with ORs of 1.67 (95 % CI 1.01-2.76) for the combined GA/AA of rs1805377 and 1.69 (95 % CI 1.03-2.77) for rs1056503 TG/GG; these associations were no longer statistically significant in multivariable conditional logistic regression models. When examining the association of SNPs with phenotype, we found that genotypes of XRCC5 rs3834 and rs1051685, which were highly correlated with each other, were associated with end-joining (EJ) capacity; women with the XRCC5 rs3834 GA genotype had better DNA repair as measured by higher levels of EJ capacity (37.8 ± 14.1 % for GA vs. 27.9 ± 11.8 % for GG carriers p = 0.0006). Women with the AA genotype of BRCA1 rs799917 also had higher EJ capacity (35.1 ± 9.2 %) than those with GG (26.4 ± 10.1 %, p = 0.02). CONCLUSIONS: Overall, we found that selected DSBR genotypes were associated with phenotype, although they were not associated with BC risk itself, suggesting that phenotypic measures are influenced by endogenous and exogenous factors across the life course and may be better markers than genotypic measures for ascertaining BC risk.

Double-strand breaks repair in lymphoblastoid cell lines from sisters discordant for breast cancer from the New York site of the BCFR.

Unrepaired DNA double-strand breaks (DSBs) may have serious consequences for cells by inducing chromosomal aberrations, thereby increasing genetic instability and cancer risk. One's capacity to repair DSB is therefore an important factor to consider when estimating cancer risk. We assessed DNA end-joining (EJ) capacity in cell lines derived from sisters discordant for breast cancer to determine if individual differences in DSB repair are a significant risk factor. We used an in vitro phenotypic assay on nuclear extracts from lymphoblasts of 179 subjects including 86 cases and 93 controls. EJ activity was functionally estimated as the ability of extracts to join together monomers of the plasmid pUC18 linearized either with sticky (EcoRI) or blunt ends (HincII). Mean percentage of EJ capacity was slightly lower in cases than controls, both for EcoRI (cases 27.9 +/- 11.1; controls 29.6 +/- 10.7, P = 0.28) and HincII substrates (cases 28.8 +/- 12.2; controls 30.6 +/- 13.0, P = 0.36); however, no significant differences were observed. Categorizing EJ capacity into tertiles and using the highest activity as the referent, we observed elevated associations for each tertile of decreased repair [Odds ratio (OR) = 2.20, 95% confidence interval (CI) = 0.77-6.22 and OR = 4.22, 95% CI thinsp;= 1.22-14.0, P = 0.02], respectively, for EcoRI. Results were not statistically significant for HincII (OR = 1.37, 95% CI = 0.51-3.70 and OR = 2.32, 95% CI = 0.57-9.38, P = 0.24). These data suggest that individual differences in EJ capacity may represent a risk factor predisposing women to breast cancer.

Use of the land snail Helix aspersa as sentinel organism for monitoring ecotoxicologic effects of urban pollution: an integrated approach.

Atmospheric pollution from vehicular traffic is a matter of growing interest, often leading to temporary restrictions in urban areas. Although guidelines indicate limits for several parameters, the real toxicologic impacts remain largely unexplored in field conditions. In this study our aim was to validate an ecotoxicologic approach to evaluate both bioaccumulation and toxicologic effects caused by airborne pollutants. Specimens of the land snail Helix aspersa were caged in five sites in the urban area of Ancona, Italy. After 4 weeks, trace metals (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) and polycyclic aromatic hydrocarbons (PAHs) were measured and these data integrated with the analyses of molecular and biochemical responses. Such biomarkers reflected the induction of detoxification pathways or the onset of cellular toxicity caused by pollutants. Biomarkers that correlated with contaminant accumulation included levels of metallothioneins, activity of biotransformation enzymes (ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase), and peroxisomal proliferation. More general responses were investigated as oxidative stress variations, including efficiency of antioxidant defenses (catalase, glutathione reductase, glutathione S-transferases, glutathione peroxidases, and total glutathione) and total oxyradical scavenging capacity toward peroxyl and hydroxyl radicals, onset of cellular damages (i.e., lysosomal destabilization), and loss of DNA integrity. Results revealed a marked accumulation of metals and PAHs in digestive tissues of organisms maintained in more traffic-congested sites. The contemporary appearance of several alterations confirmed the cellular reactivity of these chemicals with toxicologic effects of potential concern for human health. The overall results of this exploratory study suggest the utility of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas. Key words: atmospheric pollutants, bioindicators, biomarkers, DNA integrity, lysosomes, metallothioneins, oxidative stress, peroxisomes, polycyclic aromatic hydrocarbons, trace metals.

Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis.

Genotoxicity studies using the single cell gel electrophoresis (SCGE) assay indicate that basal levels of DNA strand breaks (SBs) in marine invertebrates are higher and more variable than those in marine vertebrates. This elevated level of DNA damage was attributed to a large number of alkali-labile sites, which are characteristic of the tightly-packaged DNA in invertebrate cells. To investigate if altering the SCGE protocol can artificially modulate high levels of SBs, SCGE experiments were performed on haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) using proteinase K (PK) digestion in combination with assay buffers containing various concentrations of EDTA. In addition, the effects of Trolox (soluble antioxidant) and aurintricarboxylic acid (ATA; inhibitor of Ca(2+)/Mg(2+)-dependent nucleases) also were tested. The levels of SBs in M. galloprovincialis cells were compared with SBs in cells from a terrestrial mollusk (the snail Helix aspersa), and a teleost fish (the seabass Dicentrarchus labrax). The integrity of M. galloprovincialis DNA isolated with phenol extractions using EDTA, Trolox, and ATA was further assayed by gel electrophoresis. High SBs in mussel cells were reduced by combining EDTA with PK digestion, or using Trolox or ATA during cell processing for the SCGE assay. Snails and seabass had lower levels of SBs in the SCGE assay, and the levels were not affected by the protocol modifications. Adding EDTA, Trolox, or ATA to phenol extractions of M. galloprovincialis genomic DNA also reduced the extent of DNA fragmentation. These results suggest that the internal fluids of M. galloprovincialis may increase the basal levels of DNA SBs through oxidative and/or enzyme-mediated pathways. M. galloprovincialis is used extensively as a sentinel species for assessing the genotoxic hazard of marine pollutants. Our data suggest that the SCGE protocol should be carefully considered when assessing DNA damage in these species.

Pro-oxidant effects of extremely low frequency electromagnetic fields in the land snail Helix aspersa.

Pro-oxidant effects of extremely low frequency (ELF) 50-Hz magnetic fields were investigated in the land snail Helix aspersa exposed both in short-term laboratory treatments and under field conditions by maintaining the organisms in the proximity of a power line for up to 2 months. Oxidative perturbations were investigated as individual antioxidants (catalase, glutathione reductase, glutathione S-transferases, and total glutathione) and total scavenging capacity toward peroxyl radicals and hydroxyl radicals. Accumulation of lipid peroxidation products, destabilization of lysosomal membranes, and loss of DNA integrity were also evaluated as markers of cell damage. The overall results indicated an oxidative challenge caused by ELF magnetic fields with particularly prompt and sensitive responses for catalase, glutathione reductase, and the overall capability to neutralize peroxyl radicals. Cell injuries occurred to different extents according to duration and intensity of electromagnetic exposure and confirmed complex cause-effect relationships between pro-oxidant factors, efficiency of antioxidant defenses, and the onset of oxidative toxicity. This study highlights the importance of a multimarker approach for detecting a wide panel of biological responses, the necessity of investigating the long-term effects of early oxidative responses, and the role of ELF in enhancing susceptibility to other forms of pathologies or diseases.

Immunofluorescent detection of 8-oxo-dG and PAH bulky adducts in fish liver and mussel digestive gland.

Development of methodologies to detect DNA damage induced by pollutants is of increasing concern in marine ecotoxicology. We previously described an immunoperoxidase method for revealing 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-dG) in marine organisms. In this work, the approach was extended to immunofluorescence detection and to the use of another antibody for polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Specimens of European eel (Anguilla anguilla) and Mediterranean mussel (Mytilus galloprovincialis) were exposed to benzo(a)pyrene (B[a]P), as model chemical carcinogen to induce both oxidized bases and B[a]P diol-epoxide-deoxyguanosine adducts. Cryostat sections of liver and digestive gland from both species were immunostained, and DNA damage was semiquantitatively evaluated by an image analysis system. Compared to untreated organisms, B[a]P-exposed organisms exhibited increased levels of oxidative DNA damage; in eels, which rapidly metabolize PAHs, the occurrence of B[a]P-DNA adducts was also detected. The immunofluorescent assay maintained all the advantages previously reported for the immunoperoxidase protocol. Both methods were tested on the same eel specimens and the immunofluorescence method showed a greater extent of relative DNA damage and a higher sensitivity. Although field validation is being carried out, our results indicate the utility of antibodies to rapidly detect DNA alterations in aquatic organisms, and to investigate the risk associated with genotoxins in marine environment.

Application of an immunoperoxidase staining method for detection of 7,8-dihydro-8-oxodeoxyguanosine as a biomarker of chemical-induced oxidative stress in marine organisms.

7,8-Dihydro-8-oxodeoxyguanosine (8-oxo-dG) is a typical modification of DNA caused by oxygen free radicals and can be an useful biomarker for pollutants inducing oxidative stress. An immunoperoxidase method using monoclonal antibody 1F7 toward 8-oxo-dG was applied to tissues and smeared cells of marine organisms for detection and quantification of oxidative DNA damage in such models. The assay, previously employed on human cells, was assessed for the first time on Mediterranean mussels (Mytilus galloprovincialis) and European eels (Anguilla anguilla), exposed to model pro-oxidant chemicals, namely benzo[a]pyrene (B[a]P) and copper. Quantification of 8-oxo-dG was microscopically carried out and expressed as relative nuclear staining intensity. Higher levels of oxidative DNA damage were detected in the digestive glands of treated mussels compared to controls, while the effect was less pronounced in haemocytes, characterized by more elevated basal levels of 8-oxo-dG. The assay was suitable for detection of 8-oxo-dG also in fish liver sections indicating consistent damage after B[a]P exposure. The main advantage of the immunohistochemical approach is the elimination of DNA extraction which considerably reduces the processing of biological samples. In addition, the assay requires small amounts of frozen tissues or fixed cells for detection of 8-oxo-dG and is potentially able to discriminate variable susceptibility to oxidative stress in different cell types. Although further investigations are required for the improvement and the validation of the assay in field conditions, laboratory exposures provided useful indications on the consistency of the approach and the efficacy of antibody 1F7 in marine organisms for a rapid assessment of pollutant-induced oxidative DNA damage.

Oxidative damage to DNA: an immunohistochemical approach for detection of 7,8-dihydro-8-oxodeoxyguanosine in marine organisms.

The modified nucleoside 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-dG) is an index of oxidative DNA damage. An immunohistochemical approach based on the use of monoclonal antibody 1F7 against 8-oxo-dG was investigated in marine organisms with immunoperoxidase and immunofluorescent detection. Relative staining intensity as a measure of the 8-oxo-dG level was microscopically assessed. After laboratory exposures to benzo[a]pyrene (B[a]P), higher levels of oxidative DNA damage were clearly detected in all treated animals compared to controls. While this method eliminates DNA extraction reducing the processing of biological samples, absolute values are not provided. Further, the method requires only small amounts of tissue and potentially discriminates susceptibility to oxidative damage in different cell types. These results suggest that the assay should have practical applications in marine ecotoxicology.