Formation Of Bulky DNA Adducts Research Articles

Traffic-Related Air Pollution and Ground-Level Ozone Associated Global DNA Hypomethylation and Bulky DNA Adduct Formation.

Studies have indicated that air pollution, including surface-level ozone (O3), can significantly influence the risk of chronic diseases. To better understand the carcinogenic mechanisms of air pollutants and identify predictive disease biomarkers, we examined the association between traffic-related pollutants with DNA methylation alterations and bulky DNA adducts, two biomarkers of carcinogen exposure and cancer risk, in the peripheral blood of 140 volunteers-95 traffic police officers, and 45 unexposed subjects. The DNA methylation and adduct measurements were performed by bisulfite-PCR and pyrosequencing and 32P-postlabeling assay. Airborne levels of benzo(a)pyrene [B(a)P], carbon monoxide, and tropospheric O3 were determined by personal exposure biomonitoring or by fixed monitoring stations. Overall, air pollution exposure was associated with a significant reduction (1.41 units) in global DNA methylation (95% C.I. -2.65-0.04, p = 0.026). The decrement in ALU repetitive elements was greatest in the policemen working downtown (95% C.I. -3.23--0.49, p = 0.008). The DNA adducts were found to be significantly increased (0.45 units) in the municipal officers with respect to unexposed subjects (95% C.I. 0.02-0.88, p = 0.039), mainly in those who were controlling traffic in downtown areas (95% C.I. 0.39-1.29, p < 0.001). Regression models indicated an increment of ALU methylation at higher B(a)P concentrations (95% C.I. 0.03-0.60, p = 0.032). Moreover, statistical models showed a decrement in ALU methylation and an increment of DNA damage only above the cut-off value of 30 µg/m3 O3. A significant increment of 0.73 units of IL-6 gene methylation was also found in smokers with respect to non-smokers. Our results highlighted the role of air pollution on epigenetic alterations and genotoxic effects, especially above the target value of 30 µg/m3 surface-level O3, supporting the necessity for developing public health strategies aimed to reduce traffic-related air pollution molecular alterations.

Force-Dependent Intercalative Bulky DNA Adduct Formation Detected by Single-Molecule Stretching.

Quantitatively analyzing the binding topology and reactivity is essential for understanding the cytotoxic or tumorigenic activities of bulky DNA adducts formed by chemotherapeutic drugs or carcinogens. Biochemical methods require purification of DNA and discontinuous steps to digest or label the adducts and thus have difficulties in identifying the binding topology and are not suitable for detecting unstable adducts. Herein, we used a single-molecule stretching assay to characterize the number of intercalative adducts, the formation kinetics, and the mechanical properties of intercalative DNA adducts based on measuring adduct-induced DNA elongation. We analyzed various reactive conditions, including formaldehyde-mediated anthracycline-DNA adducts, UV light-catalyzed psoralen-DNA adducts, and liver S9 fraction-catalyzed aflatoxin B1-DNA adducts. We showed that adduct formation abilities are correlated with the noncovalent intercalation binding ability. External forces on double-stranded DNA increased the intercalation of ligands and can result in a 1.8- to 5.3-fold increase in DNA adduct formation.

In Vitro Assessment of the Genotoxic Hazard of Novel Hydroxamic Acid- and Benzamide-Type Histone Deacetylase Inhibitors (HDACi).

Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies.

Formation of Bulky DNA Adducts by Non-Enzymatic Production of 1,2-Naphthoquinone-Epoxide from 1,2-Naphthoquinone under Physiological Conditions.

Quinones may be formed metabolically or abiotically from environmental pollutants and polycyclic aromatic hydrocarbons (PAHs); many are recognized as toxicological intermediates that cause a variety of deleterious cellular effects including mutagenicity. The PAH-o-quinone, 1,2-naphthoquinone (1,2-NQ), may exert its genotoxic effects through interactions with cellular nucleophiles such as DNA, however, the mechanisms of 1,2-NQ adduct formation are still under investigation. With the aim to further understand these mechanisms, the chemical structures of adducts formed from the reaction of 2'-deoxyguanosine (dG) with 1,2-NQ under physiological conditions were investigated by liquid chromatography electrospray ionization tandem mass spectrometry and 1H NMR analyses. Results showed that 1,2-NQ underwent non-enzymatic oxidation to form a 1,2-NQ-epoxide which in turn formed at least four bulky adducts with dG, and these adducts were more likely to be formed under physiological conditions. A mechanism was proposed whereby hydration of 1,2-NQ to form unstable naphthohydroquinones and 2-hydroxy-1,4-naphthoquinone resulted in formation of hydrogen peroxide that oxidized 1,2-NQ. These results suggest that the genotoxicity of 1,2-NQ may not only be caused through oxidative DNA damage and adduct formation through Michael addition but also through non-enzymatic oxidative transformation of 1,2-NQ itself to form an intermediate PAH-epoxide which covalently binds to DNA.

Abstract A10: Exposure to heterocyclic aromatic amines, genetic susceptibility and bulky DNA adduct levels in blood leukocytes

Abstract Background: There is compelling evidence to suggest that aspects of diet influence cancer risk; specifically, high meat consumption is associated with elevated risks. Exposure to heterocyclic aromatic amines (HAAs), carcinogens produced in meat when cooked at high temperatures, is a hypothesized explanation for the meat-cancer relationship. Reactive HAA metabolites form adducts with DNA; left unrepaired, adducts can induce mutations which may initiate/promote carcinogenesis. Genetic differences in the ability to biotransform HAAs (as conferred by polymorphisms in CYP1A1, CYP1A2, CYP1B1, NAT1 and NAT2) and repair DNA adducts (as conferred by polymorphisms in XPA, XPD and XRCC1) is postulated to modify the dietary HAAs-DNA adduct relationship. Methods: In a cross-sectional study of 99 healthy volunteers recruited from Kingston, Ontario, Canada, dietary exposure to HAAs and bulky DNA adduct levels in blood leukocytes was investigated. A detailed questionnaire was used in combination with a mutagen database to estimate average intake of dietary HAAs. Specifically, the detailed questionnaire obtained the average frequency of consumption, usual level of doneness and usual serving size of nine commonly consumed meats items with high HAA content. Bulky DNA adduct levels were measured in blood collected after an overnight fast using 32P-postlabelling. Least squares regression was used to examine the relationship between dietary HAA exposure and bulky DNA adduct levels in blood. Gene-diet interactions between dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair were also examined. Results: No main effects of dietary exposure to HAAs on bulky DNA adduct levels were observed. However, polymorphisms in NAT1 were found to associate with bulky DNA adduct levels. Specifically, those with the putative NAT1*10 rapid acetylator phenotype had a lower adduct level than those with the slow acetylator phenotype (p=0.02). Furthermore, having five or more ‘at-risk’ genotypes was associated with higher bulky DNA adduct levels (p=0.03). Gene-diet interactions were also observed between the NAT1*10 allele and dietary HAAs (p&amp;lt;0.05); specifically, among the slow acetylator phenotype, higher intakes of dietary HAAs were associated with an increase in DNA adduct levels compared to lower intakes. Conclusions: This study provides evidence of a biologic relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct formation. The lack of a strong independent association between dietary HAAs and DNA adducts suggests that dietary HAAs are not a large contributor to bulky DNA adducts in this Canadian population; future studies should consider relevant gene-diet interactions to clarify the potential role of HAAs in carcinogenesis. Citation Format: Vikki Ho, Sarah Peacock, Thomas E. Massey, Roger W. L. Godschalk, Frederik-Jan van Schooten, Jian Chen, Will D. King. Exposure to heterocyclic aromatic amines, genetic susceptibility and bulky DNA adduct levels in blood leukocytes. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A10.

Gene-diet interactions in exposure to heterocyclic aromatic amines and bulky DNA adduct levels in blood leukocytes.

Heterocyclic aromatic amines (HAAs), carcinogens produced in meat when cooked at high temperatures, are an emerging biologic explanation for the meat-colorectal cancer relationship. HAAs form DNA adducts; left unrepaired, adducts can induce mutations, which may initiate/promote carcinogenesis. The purpose of this research was to investigate the relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct levels. Least squares regression was used to examine the relationship between dietary HAA exposure and bulky DNA adduct levels in blood measured using (32)P-postlabeling among 99 healthy volunteers. Gene-diet interactions between dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair were also examined. No main effects of dietary HAAs on bulky DNA adduct levels was found. However, those with the putative NAT1 rapid acetylator phenotype had lower adduct levels than those with the slow acetylator phenotype (P = 0.02). Furthermore, having five or more 'at-risk' genotypes was associated with higher bulky DNA adduct levels (P = 0.03). Gene-diet interactions were observed between NAT1 polymorphisms and dietary HAAs (P < 0.05); among the slow acetylator phenotype, higher intakes of HAAs were associated with an increase in DNA adduct levels compared to lower intakes. This study provides evidence of a biologic relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct formation. However, the lack of a strong main effect of HAAs suggests that dietary HAAs are not a large contributor to bulky DNA adducts in this population; future studies should consider relevant gene-diet interactions to clarify the role of HAAs in carcinogenesis.

Comparative mutagenicity and genotoxicity of particles and aerosols emitted by the combustion of standard vs. rapeseed methyl ester supplemented bio-diesel fuels: Impact of after treatment devices: Oxidation catalyst and particulate filter

Diesel exhausts are partly responsible for the deleterious effects on human health associated with urban pollution, including cardiovascular diseases, asthma, COPD, and possibly lung cancer. Particulate fraction has been incriminated and thus largely investigated for its genotoxic properties, based on exposure conditions that are, however, not relevant for human risk assessment. In this paper, original and more realistic protocols were used to investigate the hazards induced by exhausts emitted by the combustion of standard (DF0) vs. bio-diesel fuels (DF7 and DF30) and to assess the impact of exhaust treatment devices (DOC and DPF). Mutagenicity and genotoxicity were evaluated for (1) resuspended particles (“off line” exposure that takes into account the bioavailability of adsorbed chemicals) and for (2) the whole aerosols (particles+gas phase components) under continuous flow exposure (“on line” exposure). Native particles displayed mutagenic properties associated with nitroaromatic profiles (YG1041), whereas PAHs did not seem to be involved. After DOC treatment, the mutagenicity of particles was fully abolished. In contrast, the level of particle deposition was low under continuous flow exposure, and the observed mutagenicity in TA98 and TA102 was thus attributable to the gas phase. A bactericidal effect was also observed in TA102 after DOC treatment, and a weak but significant mutagenicity persisted after DPF treatment for bio-diesel fuels. No formation of bulky DNA-adducts was observed on A549 cells exposed to diesel exhaust, even in very drastic conditions (organic extracts corresponding to 500μg equivalent particule/mL, 48h exposure). Taken together, these data indicate that the exhausts issued from the bio-diesel fuels supplemented with rapseed methyl ester (RME), and generated by current diesel engines equipped with after treatment devices are less mutagenic than older ones. The residual mutagenicity is linked to the gas phase and could be due to pro-oxydants, mainly for RME-supplemented fuels.

Xenobiotic metabolism induction and bulky DNA adducts generated by particulate matter pollution in BEAS-2B cell line: geographical and seasonal influence

Airborne particulate matter (PM) toxicity is of growing interest as diesel exhaust particles have been classified as carcinogenic to humans. However, PM is a mixture of chemicals, and respective contribution of organic and inorganic fractions to PM toxicity remains unclear. Thus, we analysed the link between chemical composition of PM samples and bulky DNA adduct formation supported by CYP1A1 and 1B1 genes induction and catalytic activities. We used six native PM samples, collected in industrial, rural or urban areas, either during the summer or winter, and carried out our experiments on the human bronchial epithelial cell line BEAS-2B. Cell exposure to PM resulted in CYP1A1 and CYP1B1 genes induction. This was followed by an increase in EROD activity, leading to bulky DNA adduct formation in exposed cells. Bulky DNA adduct intensity was associated to global EROD activity, but this activity was poorly correlated with CYPs mRNA levels. However, EROD activity was correlated with both metal and polycyclic aromatic hydrocarbon (PAH) content. Finally, principal components analysis revealed three clusters for PM chemicals, and suggested synergistic effects of metals and PAHs on bulky DNA adduct levels. This study showed the ability of PM samples from various origins to generate bulky DNA adducts in BEAS-2B cells. This formation was promoted by increased expression and activity of CYPs involved in PAHs activation into reactive metabolites. However, our data highlight that bulky DNA adduct formation is only partly explained by PM content in PAHs, and suggest that inorganic compounds, such as iron, may promote bulky DNA adduct formation by supporting CYP activity.

The GSTM1null (deletion) and MGMT84 rs12917 (Phe/Phe) haplotype are associated with bulky DNA adduct levels in human leukocytes

Tobacco smoke and air pollutants contain carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and tobacco specific nitrosamines (TSNA), that are substrates of metabolizing enzymes generating reactive metabolites that can bind to DNA. Variation in the activity of these enzymes may modify the extent to which these metabolites can interact with DNA. We compared the levels of bulky DNA adducts in blood leukocytes from 93 volunteers living in Mexico City with the presence of 13 single nucleotide polymorphisms (SNPs) in genes related to PAH and TSNA metabolism (AhR rs2044853, CYP1A1 rs1048943, CYP1A1 rs1048943, CYP1A1 rs1799814, EPHX1 rs1051740, EPHX1 rs2234922, GSTM1 null, GSTT1 null and GSTP1 rs947894), DNA repair (XRCC1 rs25487, ERCC2 rs13181 and MGMT rs12917) and cell cycle (TP53 rs1042522). 32P-postlabeling analysis was used to quantify bulky DNA adduct formation. Genotyping was performed using PCR-RFLP. The mean levels of bulky DNA adducts were 8.51±3.66 adducts/108 nucleotides (nt) in smokers and 8.38±3.59 adducts/108 nt in non-smokers, being the difference not statistically significant. Without taking into account the smoking status, GSTM1null individuals had a marginally significant lower adduct levels compared with GSTM1 volunteers (p=0.0433) and individuals heterozygous for MGMT Leu/Phe had a higher level of bulky adducts than those who were homozygous wild type (p=0.0170). A multiple regression analysis model showed a significant association between the GSTM1 (deletion) and MGMT rs12917 (Phe/Phe) haplotype and the formation of DNA adducts in smokers (R2=0.2401, p=0.0215). The presence of these variants conferred a greater risk for higher adduct levels in this Mexican population.

MATERNAL, DIETARY AND ENVIRONMENTAL PREDICTORS OF BULKY DNA ADDUCTS IN CORD BLOOD: A EUROPEAN MOTHER–CHILD STUDY (NEWGENERIS).

Background: Bulky DNA adducts in cord blood reflect prenatal effective dose of exposure to genotoxic compounds such as polycyclic aromatic hydrocarbons, provide a reliable biomarker of genotoxic damage and possibly cancer risk. Little is known on sources of exposures resulting in these adducts particularly in neonates. Aims: We examined maternal, dietary and environmental predictors of bulky DNA adduct levels in neonates to better understand our previous finding of an association between higher adduct levels and lower birth weight. Methods: Pregnant women from Greece, Spain, England, Denmark and Norway were recruited in 2006-2010. Adducts were measured by the 32P-postlabelling technique in cord white blood cells (n=511). Maternal diet and other personal characteristics were assessed via questionnaires. Land-use regression models were used to estimate individual exposure to air pollution. Results: Adduct levels tended to be higher in neonates born to women who smoked as compared with those of non-smokers (10.0 (n=81) vs. 7.6 (n=424) adducts per 108 nucleotide, p=0.10). Spanish and Greek neonates had higher adduct levels than the Northern Europeans (12.7 (n=237) vs. 7.0 (n=373) adducts per 108 nucleotides, p&lt;0.001). Neonates born during June-August had the highest adduct levels. Higher maternal pre-pregnancy body-mass index, births during June-August, and high maternal intake of cured and smoked meat was associated with higher adduct levels (p&lt;0.05). A protective effect of maternal intake of fruits rich in vitamin C was also found (p=0.03). Ambient air pollution (PM2.5) at the home address during pregnancy was not associated with adduct levels in Greece and England. Estimation of air pollution is ongoing in the other countries and a complete analysis will be presented. Conclusions: These findings suggest that prenatal exposures from maternal lifestyle and so far unknown country-specific factors influence the bulky DNA adduct formation in neonates. Acknowledgement: NewGeneris and ESCAPE (EU FOOD-CT-2005-016320 and 211250).

Bulky DNA Adducts in Cord Blood, Maternal Fruit-and-Vegetable Consumption, and Birth Weight in a European Mother–Child Study (NewGeneris)

Background: Tobacco-smoke, airborne, and dietary exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with reduced prenatal growth. Evidence from biomarker-based studies of low-exposed populations is limited. Bulky DNA adducts in cord blood reflect the prenatal effective dose to several genotoxic agents including PAHs.Objectives: We estimated the association between bulky DNA adduct levels and birth weight in a multicenter study and examined modification of this association by maternal intake of fruits and vegetables during pregnancy.Methods: Pregnant women from Denmark, England, Greece, Norway, and Spain were recruited in 2006–2010. Adduct levels were measured by the 32P-postlabeling technique in white blood cells from 229 mothers and 612 newborns. Maternal diet was examined through questionnaires.Results: Adduct levels in maternal and cord blood samples were similar and positively correlated (median, 12.1 vs. 11.4 adducts in 108 nucleotides; Spearman rank correlation coefficient = 0.66, p < 0.001). Cord blood adduct levels were negatively associated with birth weight, with an estimated difference in mean birth weight of –129 g (95% CI: –233, –25 g) for infants in the highest versus lowest tertile of adducts. The negative association with birth weight was limited to births in Norway, Denmark, and England, the countries with the lowest adduct levels, and was more pronounced in births to mothers with low intake of fruits and vegetables (–248 g; 95% CI: –405, –92 g) compared with those with high intake (–58 g; 95% CI: –206, 90 g)Conclusions: Maternal exposure to genotoxic agents that induce the formation of bulky DNA adducts may affect intrauterine growth. Maternal fruit and vegetable consumption may be protective.Citation: Pedersen M, Schoket B, Godschalk RW, Wright J, von Stedingk H, Törnqvist M, Sunyer J, Nielsen JK, Merlo DF, Mendez MA, Meltzer HM, Lukács V, Landström A, Kyrtopoulos SA, Kovács K, Knudsen LE, Haugen M, Hardie LJ, Gützkow KB, Fleming S, Fthenou E, Farmer PB, Espinosa A, Chatzi L, Brunborg G, Brady NJ, Botsivali M, Arab K, Anna L, Alexander J, Agramunt S, Kleinjans JC, Segerbäck D, Kogevinas M. 2013. Bulky DNA adducts in cord blood, maternal fruit-and-vegetable consumption, and birth weight in a European mother–child study (NewGeneris). Environ Health Perspect 121:1200–1206; http://dx.doi.org/10.1289/ehp.1206333

Association studies of excision repair cross-complementation group 1 ( ERCC1) haplotypes with lung and head and neck cancer risk in a Caucasian population

Background: The formation of bulky DNA adducts caused by diol epoxide derivatives of polycyclic aromatic hydrocarbons has been associated with tobacco-induced cancers, and inefficient repair of such adducts by the nucleotide excision repair (NER) system has been linked to increased risk of tobacco-induced lung and head and neck (H&N) cancers. The human excision repair cross-complementation group 1 (ERCC1) protein is essential for a functional NER system and genetic variation in ERCC1 may contribute to impaired DNA repair capacity and increased lung and H&N cancer risk. Methods: In order to comprehensively capture common genetic variation in the ERCC1 gene, Caucasian data from the International HapMap project was used to assess linkage disequilibrium and choose four tagSNPs (rs1319052, rs3212955, rs3212948, and rs735482) in the ERCC1 gene to genotype 452 lung cancer cases, 175 H&N cancer cases, and 790 healthy controls. Haplotypes were estimated using expectation maximization (EM) algorithm, and haplotype association with cancer was investigated using Haplo.stats software adjusting for known covariates. Results: The genotype and haplotype frequencies matched previous estimates from Caucasians. There was no significant difference in the prevalence of rs1319052, rs3212955, rs3212948, and rs735482 when comparing lung or H&N cancer cases with controls ( p-values > 0.05). Similarly, there was no association between ERCC1 haplotypes and lung or H&N cancer susceptibility in this Caucasian population ( p-values > 0.05). No associations were found when stratifying lung cancer cases by histology, sex, smoking status, or smoking intensity. Conclusions: This study suggests that ERCC1 polymorphisms and haplotypes do not play a role in lung and H&N cancer susceptibility in Caucasians.

977 THIOTHYMIDINE COMBINED WITH UVA AS A POTENTIAL NOVEL THERAPY FOR BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2010977 THIOTHYMIDINE COMBINED WITH UVA AS A POTENTIAL NOVEL THERAPY FOR BLADDER CANCER Simon Pridgeon, Gordon Taylor, Keiran O'Toole, Mary Robinson, and Alan Boddy Simon PridgeonSimon Pridgeon London, United Kingdom More articles by this author , Gordon TaylorGordon Taylor Newcastle upon Tyne, United Kingdom More articles by this author , Keiran O'TooleKeiran O'Toole Newcastle upon Tyne, United Kingdom More articles by this author , Mary RobinsonMary Robinson Newcastle upon Tyne, United Kingdom More articles by this author , and Alan BoddyAlan Boddy Newcastle upon Tyne, United Kingdom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1924AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Thiothymidine (TT) is a thymidine analogue which is readily incorporated into cellular DNA. TT is sensitive to UVA (336nm) and on exposure undergoes photochemichal reactions. It is hypothesised that TT incorporated into bladder tumors and subsequently exposed to UVA will form cytotoxic DNA lesions. METHODS MYU-3L and AY27 bladder cancer cell lines were cultured with TT and exposed to UVA. Cytotoxicity was assessed using the SRB assay. Following DNA extraction and digestion, TT incorporation was measured using LC/MS. DNA lesions were characterised using the COMET assay. Bladder tumours were established in Fischer rats by intravesical instillation of tumor cells. TT incorporation into tumor DNA following intravesical or intravenous administration was measured using LC/MS. In vivo efficacy was studied in 5 animals treated with TT and UVA delivery using a 0.4 mm fiber optic. TT uptake into human bladder tumours was studied in an ex-vivo setting. RESULTS TT was measured in DNA of bladder cancer cells replacing 0.1-0.6% thymidine. 100% growth inhibition was observed when AY27 cell lines were incubated with 2uM TT and exposure to 2.5 KJ/m2 UVA. For MYU-3L cells, 100% growth inhibition was observed following incubation with 20 uM TT and exposure to 10 KJ/m2 UVA. Following treatment with TT and UVA, the COMET assay demonstrated the formation of bulky DNA adducts with >50% reduction in Olive tail moments. ProteinaseK treatment suggested these DNA lesions were both DNA-DNA adducts and DNA-protein crosslinks. TT and UVA treatment was associated with an 8-fold increase in caspase 3/7 activity and positive annexin binding suggesting apoptosis. Bladder tumours were successfully established in Fischer rats using MYU-3L cells. TT incorporation was detectable follwing both intravesical and intravenous TT administration. TT replaced 0.0035% of thymidine residues following a 2 hour intravesical TT instillation (16mg) and 0.2 % thymidine replacement was measure following IV TT injection (32 mg). Treatment responses were observed in 60% rats following intravenous TT and intravesical UVA (5 KJ/m2) delivery. TT was detected in 30/40 human bladder tumor resection specimens following 24 hour ex-vivo incubation replacing 0.03 % thymidine. CONCLUSIONS TT has been shown to be incorporated into bladder cancer cell lines. The subsequent exposure to low doses of UVA results in growth inhibition due to the formation of DNA crosslinks. These crosslinks are associated with apoptosis. In vivo and ex vivo treatment suggest that the combination of TT and UVA may offer a novel selective local treatment for bladder cancer © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e380 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Simon Pridgeon London, United Kingdom More articles by this author Gordon Taylor Newcastle upon Tyne, United Kingdom More articles by this author Keiran O'Toole Newcastle upon Tyne, United Kingdom More articles by this author Mary Robinson Newcastle upon Tyne, United Kingdom More articles by this author Alan Boddy Newcastle upon Tyne, United Kingdom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Aromatic DNA adducts and polymorphisms in metabolic genes in healthy adults: findings from the EPIC-Spain cohort

Aromatic compounds such as polycyclic aromatic hydrocarbons, arylamines and heterocyclic amines require metabolic activation to form metabolites able to bind to DNA, a process mediated by polymorphic enzymes. We measured aromatic DNA adducts in white blood cells by the (32)P-post-labelling assay in a sample of 296 healthy adults (147 men and 149 women) from five regions of Spain. We also analyzed functional polymorphisms in the metabolic genes CYP1A1, CYP1A2, EPHX1, GSTM1, GSTT1, NAT2 and SULT1A1. A significant increased level of DNA aromatic adducts was found related to the fast oxidation-hydrolysis phenotype defined by the polymorphism I462V in CYP1A1, the allele A in IVS1-154C>A of CYP1A2 and the combination Tyrosine-Arginine for Y113H and H139R of EPHX1. Geometric means (adducts per 10(-9) normal nucleotides) were 2.17, 4.04 and 6.30 for slow, normal and fast phenotypes, respectively (P-trend = 0.01). Slow acetylation by NAT2 was associated with a significant decrease in adduct level; subjects with slow alleles *5A and *7A/B had in average 1.56 x 10(-9)adducts, as compared with 5.60 for those with normal NAT2 activity (P-value = 0.01). No association was seen with polymorphisms of other metabolic genes such as GSTM1, GSTT1 or SULT1A1. We concluded that the metabolic pathways of oxidation, hydrolysis and acetylation are relevant to the formation of bulky DNA adducts. This could suggest a potential involvement of aromatic compounds in the formation of such adducts; however, given lack of specificity of the post-labeling assay, a firm conclusion cannot be drawn.

Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM 2.5) in human lung epithelial A549 cells

To improve the knowledge of the underlying mechanisms of action involved in air pollution Particulate Matter (PM)-induced toxicity in human lungs, with a particular interest of the crucial role played by coated-organic chemicals, we were interested in the metabolic activation of Polycyclic Aromatic Hydrocarbons (PAH)-coated onto air pollution PM, and, thereafter, the formation of PAH–DNA adducts in a human lung epithelial cell model (A549 cell line). Cells were exposed to Dunkerque city’s PM 2.5 at its Lethal Concentrations at 10% and 50% (i.e. LC 10 = 23.72 μg/mL or 6.33 μg/cm 2, and LC 50 = 118.60 μg/mL or 31.63 μg/cm 2), and the study of Cytochrome P450 (CYP) 1A1 gene expression (i.e. RT-PCR) and protein activity (i.e. EROD activity), and the formation of PAH–DNA adducts (i.e. 32P-postlabeling), were investigated after 24, 48, and/or 72 h. PAH, PolyChlorinated Dibenzo- p-Dioxins and -Furans (PCDD/F), Dioxin-Like PolyChlorinated Biphenyls (DLPCB), and PolyChlorinated Biphenyls (PCB)-coated onto collected PM were determined (i.e. GC/MS and HRGC/HRMS, respectively), Negative (i.e. TiO 2 or desorbed PM, dPM; EqLC 10 = 19.42 μg/mL or 5.18 μg/cm 2, and EqLC 50 = 97.13 μg/mL or 25.90 μg/cm 2), and positive (i.e. benzo( a)pyrene; 1 μM) controls were included in the experimental design. Statistically significant increases of CYP1A1 gene expression and protein activity were observed in A549 cells, 24, 48 and 72 h after their exposure to dPM, suggesting thereby that the employed outgassing method was not efficient enough to remove total PAH. Both the CYP1A1 gene expression and EROD activity were highly induced 24, 48 and 72 h after cell exposure to PM. However, only very low levels of PAH–DNA adducts, also not reliably quantifiable, were reported 72 h after cell exposure to dPM, and, particularly, PM. The relatively low levels of PAH together with the presence of PCDD/F, DLPCB, and PCB-coated onto Dunkerque City’s PM 2.5 could notably contribute to explain the borderline detection of PAH–DNA adducts in dPM and/or PM-exposed A549 cells. Hence, remaining very low doses of PAH in dPM or relatively low doses of PAH-coated onto PM were involved in enzymatic induction, a key feature in PAH-toxicity, but failed to show a clear genotoxicity in this in vitro study. We also concluded that, in the human lung epithelial cell model we used, and in the experimental conditions we chose, bulky-DNA adduct formation was apparently not a major factor involved in the Dunkerque City’s PM 2.5-induced toxicity.

Bulky DNA Adduct Formation and Risk of Bladder Cancer

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with risk of bladder cancer and with increased bulky DNA adduct levels in several studies, mainly in smokers. We investigated the relation between bulky PAH-DNA adducts in peripheral blood mononuclear cells and bladder cancer in nonsmoking subjects from a large hospital-based case-control study in Spain. Additionally, we examined the association between DNA adduct formation and several air pollution proxies. The study comprised 76 nonsmoking cases and 76 individually matched controls by sex, region of residence, age, and smoking status (never, former). To maximize the relevance of the DNA adduct measurement as a proxy of PAH exposure, subjects selected had not changed residence, occupation, and major lifestyle factors during the last 10 years. Bulky DNA adducts were measured using the (32)P-postlabeling technique, nuclease P1 treatment. The percentage of detectable adducts was higher in controls (41%) than in cases (32%) with an odds ratio of 0.75 (95% confidence interval, 0.36-1.58). In an analysis limited to controls, a higher percentage of DNA adducts was found among those whose last residence was in a big city (50%) compared with those living in villages (19%; P = 0.04). No consistent associations were found for other markers of air pollution. In this study, among nonsmokers with stable environmental and lifestyle factors, bulky DNA adducts were not associated with bladder cancer risk. Results do not support an association of bladder cancer risk with low-level exposure to PAHs as measured through the formation of bulky DNA adducts in peripheral mononuclear cells.

Molecular and Cytogenetical Alterations Induced by Environmental Cigarette Smoke in Mice Heterozygous for Fhit

Previous studies in humans and animal models provided evidence that the Fhit gene is an early target for cigarette smoke. We compared the induction of a variety of molecular and cytogenetical alterations in B6-129(F(1)) mice, either wild type or Fhit(+/-), after whole-body exposure to environmental cigarette smoke (ECS) for 15 consecutive days. Both mouse genotypes responded to ECS with a loss of Fhit protein in the bronchial epithelium, accompanied by induction of apoptosis and stimulation of cell proliferation. ECS induced formation of bulky DNA adducts in whole lung. In addition, ECS caused cytogenetical damage both in the respiratory tract and at a systemic level, as shown by a significant increase of micronucleus frequency in pulmonary alveolar macrophages, bone marrow polychromatic erythrocytes, and peripheral blood normochromatic erythrocytes of both wild-type and Fhit(+/-) mice. These results are compared with those generated in other species, strains, and genotypes of rodents exposed to ECS that we investigated previously. Although the loss of Fhit protein in the bronchial epithelium of ECS-exposed B6-129(F(1)) mice provides further evidence that the Fhit gene is an early molecular target for ECS, heterozygosity for Fhit does not seem to confer an increased susceptibility of mice in terms of the investigated early biomarkers.

401 POSTER Pharmacokinetic characterization of BI 2536 - a novel Plk1 inhibitor - in advanced cancer patients

Tobacco smoke and air pollutants contain carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and tobacco specific nitrosamines (TSNA), that are substrates of metabolizing enzymes generating reactive metabolites that can bind to DNA. Variation in the activity of these enzymes may modify the extent to which these metabolites can interact with DNA. We compared the levels of bulky DNA adducts in blood leukocytes from 93 volunteers living in Mexico City with the presence of 13 single nucleotide polymorphisms (SNPs) in genes related to PAH and TSNA metabolism (AhR rs2044853, CYP1A1 rs1048943, CYP1A1 rs1048943, CYP1A1 rs1799814, EPHX1 rs1051740, EPHX1 rs2234922, GSTM1 null, GSTT1 null and GSTP1 rs947894), DNA repair (XRCC1 rs25487, ERCC2 rs13181 and MGMT rs12917) and cell cycle (TP53 rs1042522). 32P-postlabeling analysis was used to quantify bulky DNA adduct formation. Genotyping was performed using PCR-RFLP. The mean levels of bulky DNA adducts were 8.51 ± 3.66 adducts/108 nucleotides (nt) in smokers and 8.38 ± 3.59 adducts/108 nt in non-smokers, being the difference not statistically significant. Without taking into account the smoking status, GSTM1null individuals had a marginally significant lower adduct levels compared with GSTM1 volunteers (p = 0.0433) and individuals heterozygous for MGMT Leu/Phe had a higher level of bulky adducts than those who were homozygous wild type (p = 0.0170). A multiple regression analysis model showed a significant association between the GSTM1 (deletion) and MGMT rs12917 (Phe/Phe) haplotype and the formation of DNA adducts in smokers (R2 = 0.2401, p = 0.0215). The presence of these variants conferred a greater risk for higher adduct levels in this Mexican population.

DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas : Potential use for genotoxicant biomonitoring of fresh water ecosystems

The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543–1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 μg g −1 dry weight) in comparison to individuals from the reference site (0.053 μg g −1 dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10 8 nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 μg g −1 dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[ a]pyrene led to a dose-dependent accumulation of B[ a]P (up to 7063 μg g −1 dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10 8 nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[ a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable biomarker to monitor PAH-exposure and evaluate genotoxicity in fresh water ecosystems.

Modulation of multigene expression and proteome profiles by chemopreventive agents

Analysis of transcriptome and proteome profiles by microarray technologies provides a formidable, new tool in cancer chemoprevention research. An ideal chemopreventive agent should not excessively alter per se the basal make-up of multigene expression and protein synthesis and should at the same time be able to attenuate alterations induced by risk factors. In order to validate this working hypothesis, we previously performed a series of studies in animal models using the thiol N-acetyl- l-cysteine (NAC) and the nonsteroidal antiinflammatory drug sulindac. We report herein the results of new studies evaluating modulation of DNA adduct levels and expression of 4858 genes in lung and liver of Sprague-Dawley rats, either unexposed or exposed to environmental cigarette smoke (ECS). The tested chemopreventive agents included NAC, oltipraz (OPZ), 5,6-benzoflavone (5,6-BF), phenethyl isothiocyanate (PEITC), and indole 3-carbinol (I3C). Combinations of OPZ with NAC and of PEITC with I3C were also assayed. Excepting OPZ, all treatments inhibited by at least 50% the formation of bulky DNA adducts in the lung of ECS-exposed rats. Hierarchical cluster analysis and principal component analysis allowed us to classify the agents according to their influence on basal gene expression and their ability to attenuate ECS-induced transcriptome alterations. PEITC and I3C were the most effective but the least safe agents. 5,6-BF displayed intermediate patterns. OPZ was poorly effective in lung and considerably altered the basal gene expression in liver. NAC had a medium efficacy and was the safest agent, as also supported by the analysis of 518 proteins in rat lung.