Higher Adduct Levels Research Articles

Elevated polycyclic aromatic hydrocarbon-DNA adducts in benign prostate and risk of prostate cancer in African Americans.

Carcinogen-DNA adducts, a marker of DNA damage, are capable of inducing mutations in oncogenes and tumor suppressor genes, resulting in carcinogenesis. We have shown previously that polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels in prostate cancer cases vary by cellular histology and that higher adduct levels are associated with biochemical recurrence. A nested case-control study was conducted in a historical cohort of 6692 men with histopathologically benign prostate specimens. PAH-DNA adduct levels were determined by immunohistochemistry in benign prostate specimens from 536 prostate cancer case-control pairs (59% White and 41% African American). We estimated the overall and race-stratified risk of subsequent prostate cancer associated with higher adduct levels. Prostate cancer risk for men with elevated adduct levels (defined as greater than control group median) was slightly increased [odds ratio (OR) = 1.28, 95% confidence interval (CI) = 0.98-1.67, P = 0.07]. After race stratification, elevated adduct levels were significantly associated with increased risk in African American men (OR = 1.56, CI = 1.00-2.44, *P = 0.05) but not White men (OR = 1.14, CI = 0.82-1.59, P = 0.45). Elevated PAH-DNA adduct levels were significantly associated with 60% increased risk of prostate cancer among cases diagnosed 1-4 years after cohort entry (OR = 1.60, CI = 1.07-2.41) with a greater risk observed in African Americans within the first 4 years of follow-up (OR = 4.71, CI = 1.97-11.26, ***P = 0.0005). Analyses stratified by age or tumor grade revealed no additional significant heterogeneity in risk. Increased prostate cancer risk associated with high PAH-DNA adduct levels in benign prostate was found only in African Americans; risk was greatest within 4 years of follow-up, possibly reflecting a carcinogenic process not yet histologically detectable.

Acetaminophen protein adduct formation following low-dose acetaminophen exposure: comparison of immediate-release vs extended-release formulations

Acetaminophen (APAP) protein adducts are a biomarker of APAP metabolism, reflecting oxidation of APAP and generation of the reactive metabolite N-acetyl-p-benzoquinone imine. High levels of adducts correspond to liver toxicity in patients with APAP-related acute liver failure. Adduct formation following low-dose exposure to APAP has not been well studied. APAP protein adducts were measured in blood samples collected from fasted individuals who participated in a crossover study of APAP (80 mg/kg) comparing extended release (ER) and immediate release (IR) formulations. Adducts were quantified in all postdose blood samples using a validated high-performance liquid chromatography electrochemical detection (HPLC-EC) assay. Comparison of pharmacokinetic parameters for adducts did not reveal significant differences between ER and IR formulations, with one exception. Formation rates for adducts were faster for IR than the ER formulation (0.420 ± 0.157 vs. 0.203 ± 0.080 1/h), respectively. Maximum plasma concentrations (Cmax) of adducts for IR and ER were 0.108 (±0.020) and 0.100 (±0.028) nmol/ml serum, respectively, and were two orders of magnitude lower than adduct levels previously reported in adults with acute liver failure secondary to APAP. APAP protein adducts are rapidly formed following nontoxic ingestion of APAP at levels significantly lower than those associated with acute liver failure.

BPDE-like DNA adduct level in oral tissue may act as a risk biomarker of oral cancer

ObjectiveMost reports have shown that PAH-related DNA adducts are positively correlated with the smoking status of oral cancer patients. However, these reports did not focus on a specific carcinogen in cigarette smoke. The purpose of this study was to elucidate the role of the BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene)–DNA adduct in the development of oral cancer in Taiwanese patients. DesignWe enrolled 158 oral cancer patients and 64 non-cancer controls to investigate whether there were differences in susceptibility to cigarette smoke exposure in the formation of DNA adducts between cancer patients and controls. Immunohistochemistry and ELISA (enzyme-linked immunosorbent assay) were used to evaluate BPDE–DNA adduct levels in this study. ResultsOur data showed that the BPDE–DNA adduct levels were positively correlated with gender, smoking status, betel nut chewing and alcohol consumption. The difference in DNA adduct levels could be explained by genetic polymorphisms of glutathione S-transferase M1 (GSTM1), but not by cytochrome P-4501A1 (CYP1A1). Patients with high DNA adduct levels (≧34.03 adducts/108 nucleotides) had an approximately 9.936-fold risk of oral cancer compared with those with low DNA adduct levels (<34.03 adducts/108 nucleotides) (p<0.001). ConclusionsWe suggest that genetic background and carcinogen exposure may increase the risk of developing oral cancer.

Correlation between Base-Excision Repair Gene Polymorphisms and Levels of In-Vitro BPDE–Induced DNA Adducts in Cultured Peripheral Blood Lymphocytes

In vitro benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in cultured peripheral lymphocytes have been shown to be a phenotypic biomarker of individual’s DNA repair phenotype that is associated with cancer risk. In this study, we explored associations between genotypes of base-excision repair genes (PARP1 Val762Ala, APEX1 Asp148Glu, and XRCC1 Arg399Gln) and in vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes in 706 cancer-free non-Hispanic white subjects. We found that levels of BPDE-induced DNA adducts were significantly higher in ever smokers than in never smokers and that individuals with the Glu variant genotypes (i.e., Asp/Glu and Glu/Glu) exhibited lower levels of BPDE-induced DNA adducts than did individuals with the common Asp/Asp homozygous genotype (median RAL levels: 32.0 for Asp/Asp, 27.0 for Asp/Glu, and 17.0 for Glu/Glu, respectively; P trend = 0.030). Further stratified analysis showed that compared with individuals with the common APEX1-148 homozygous Asp/Asp genotype, individuals with the APEX1-148Asp/Glu genotype or the Glu/Glu genotype had a lower risk of having higher-level adducts (adjusted OR = 0.60, 95% CI: 0.36–0.98 and adjusted OR = 0.47, 95% CI: 0.26–0.86, respectively; P trend = 0.012) among smokers. Such an effect was not observed in non-smokers. However, there was no significant interaction between the APEX1 Asp148Glu polymorphism and smoking exposure in this study population (P = 0.512). Additional genotype-phenotype analysis found that the APEX1-148Glu allele had significantly increased expression of APEX1 mRNA in 270 Epstein-Barr virus-transformed lymphoblastoid cell lines, which is likely associated with more active repair activity. Our findings suggest that the functional APEX1-148Glu allele is associated with reduced risk of having high levels of BPDE-induced DNA adducts mediated with high levels of mRNA expression.

Abstract LB-63: (S)-N′-nitrosonornicotine, a constituent of smokeless tobacco, is a potent oral tumorigen in rats

Abstract Smokeless tobacco products, consisting most commonly of moist snuff placed in the mouth, either directly or in sachets, are gaining popularity in the U.S. In 2009, 7.0% of men and 11.0% of male high school students were current users. While smokeless tobacco use is unquestionably less harmful than cigarette smoking, it is nevertheless a recognized cause of oral cancer. A quantitatively important carcinogen in all smokeless tobacco products is N′-nitrosonornicotine (NNN), occurring at levels ranging from 0.4 - 17 µg/g dry weight in current products consumed in the U.S. These amounts are far higher than those of nitrosamines in other consumer products. NNN has a chiral center at its 2′-position and consequently exists as enantiomers. The major enantiomer in tobacco products is (S)-NNN. Racemic NNN, administered to F-344 rats in the drinking water, is known to induce tumors of the esophagus in rats, but there are no reports in the literature on the carcinogenicity of (S)-NNN. Based on DNA binding studies of (S)-NNN that demonstrated relatively high adduct levels in both the rat esophagus and oral cavity, we initiated a carcinogenicity study. Groups of 24 male F-344 rats, 7 weeks of age, were treated with (S)-NNN or (R)-NNN (15 ppm in the drinking water) or racemic NNN (15 rats, 30 ppm), or were given tap water. All rats in the groups treated with (S)-NNN or racemic NNN began losing weight after one year of treatment and had died or were euthanized for humane reasons by 17 months of treatment, while the rats given (R)-NNN and the controls were terminated at 20 months. Tumors were counted blinded to treatment. Necropsy of 20 (S)-NNN treated rats demonstrated a 100% incidence of oral tumors and a total of 91 oral tumors, including tumors of the tongue (1.5 tumors per rat), buccal mucosa (1.0), soft palate (0.5), and pharynx (0.75) in addition to esophageal tumors in all rats (6.1 tumors per rat). Some of the oral tumors were &amp;gt;4 mm in size: tongue (9 tumors); buccal mucosa (2); soft palate (4); pharynx (5). (R)-NNN induced oral tumors in only 5 of 24 rats and esophageal tumors in 3, while racemic NNN was also highly active causing 153 esophageal tumors and 96 oral tumors in 12 necropsied rats. Preliminary histopathological analysis of the tumors indicated that they encompass a spectrum from benign squamous papillomas to malignant squamous cell carcinomas. These results clearly demonstrate, for the first time, the strong carcinogenicity of (S)-NNN in the rat oral cavity. Thus, (S)-NNN is the only potent oral carcinogen identified in smokeless tobacco. There is an urgent need to eliminate this powerful carcinogen from tobacco products. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-63. doi:1538-7445.AM2012-LB-63

Abstract 5739: Characterization of the metabolism and DNA adduct formation of 2-amino-9H-pyrido[2,3-b]indole in human hepatocytes

Abstract 2-Amino-9H-pyrido[2-3-b]indole (AαC) is a heterocyclic aromatic amine (HAA) and considered to be a possible human carcinogen by the International Agency for Research on Cancer (IARC). Like other HAAs, AαC is metabolized in the liver and can undergo bioactivation to produce highly electrophilic derivatives that can form DNA adducts by covalent binding to DNA. Among the HAAs, AαC is of particular interest because it is present at high levels in tobacco smoke and ubiquitous in the environment. AαC was shown to form high levels of DNA adducts in primary cultured human hepatocytes. The adduct levels were higher than those formed by other HAAs such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and comparable to those derived from 4-aminobiphenyl (4-ABP), a known human carcinogen. An understanding of the metabolism of AαC and its potential to form DNA adducts would provide a better assessment of the human health risk posed by AαC. Indeed, the metabolism pathways of MeIQx, PhIP and AαC are different. These differences in metabolic processing of HAAs can lead to variable levels of DNA adducts formed in humans and impact the interindividual susceptibility towards these procarcinogens. Knowledge about the metabolism of AαC in humans is unknown. We have analyzed the role of human cytochromes P450, especially CYP1A2, in the metabolism of AαC and DNA adduct formation. The ring-oxidized 3-HO-AαC and 6-HO-AαC sulfate and glucuronide conjugates, AαC-N2-Gl, N2-acetyl-AαC and the glucuronide conjugates of 2-hydroxyamino-9H-pyrido[2,-3-b]indole (HONH-AαC) and the deoxyguanosine DNA adduct, dG-C8-AαC, were identified and quantified by liquid chromatography coupled with multistage mass spectrometry in human hepatocytes in primary culture. The levels of DNA adducts formed after a 24hr-treatment with AαC were (2 folds) lower when cells were pre-treated with furafylline, a specific CYP1A2 inhibitor; however, the levels of most of the metabolites formed were not significanly affected by furafylline. Our data suggests that CYPs other than CYP1A2 may be involved in AαC metabolism, while CYP1A2 is the main enzyme involved in the production of reactive metabolites leading to the formation of DNA adducts. The identification of CYPs and Phase II enzymes involved in AαC metabolism in human hepatocytes are under study to fully understand the health risk of AαC to humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5739. doi:1538-7445.AM2012-5739

Abstract 4118: Hypoxia affects the metabolic activation and detoxification of the environmental mutagen benzo(a)pyrene

Abstract Hypoxia is characteristic for a number of pathologies and promotes genetic instability, as exposure to low oxygen levels results in an elevated mutation frequency. We have previously shown that activation of the hypoxia responsive transcription factor HIF-1α, in the presence of sufficient oxygen, enhanced mutation frequencies induced by the environmental mutagen benzo[a]pyrene (BaP). Since proper carcinogen metabolism depends on oxygen we examined how varying oxygen levels influenced BaP metabolism by studying its activation and deactivation. The human lung carcinoma cell line A549 was treated with BaP under different oxygen concentrations (20%, 5%, and 0.2%) for 18 hours and alterations in BaP-metabolism were determined. First, we analyzed the BaP induced expression of key metabolic enzymes; expression levels of CYP1A1 and CYP1B1 was higher under hypoxia, while expression of the detoxifying enzymes UGT1A6 and UGT2B7 were significantly reduced by hypoxia. Then, several BaP metabolites in supernatants were determined by HPLC-fluorescence detection. In general, low oxygen levels lead to slower metabolism of BaP leaving more of the parent molecule intact. At the same time, BaP-7,8-dihydrodiol, the pre-cursor metabolite of the reactive metabolite BaP-7,8-dihydroxy-9,10-epoxide (BPDE), was formed in higher concentrations under hypoxia. Finally, under hypoxia DNA adducts accumulated over a period of 168 hours, whereas DNA adducts were efficiently removed under 20% oxygen, eventually leading to 1.6 times higher adduct levels under hypoxic conditions. These data indicate that the metabolism has shifted towards increased activation of BaP instead of detoxification under hypoxic conditions. These data support the idea that modulation of carcinogen metabolism may be an important additional mechanism for the observed HIF1 mediated genetic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4118. doi:1538-7445.AM2012-4118

Severe Adverse Reactions to Dental Local Anaesthetics

In the article by Sambrook et al.,1 articaine is wrongly blamed to be a risk factor for methaemoglobinaemia in humans. In the article by Wilburn-Goo and Lloyd,2 cited as reference 18 by Sambrook et al., articaine is not even mentioned at all. Higher doses of articaine than commonly administered in dental practice did not increase methaemoglobin in humans undergoing liposuction.3, 4 Besides, prilocaine which is without any doubt the local anaesthetic with the highest potency to induce methaemoglobinaemia, the second amide local anaesthetic mentioned by Wilburn-Goo and Lloyd2 is lidocaine. This is in line with conclusions from an extensive review of local anaesthetics that metabolize to 2,6-xylidine (bupivacaine, etidocaine, lidocaine, mepivacaine and ropivacaine) or o-toluidine (prilocaine).5 These two metabolites of amide local anaesthetics have a carcinogenic potential. Whereas 2,6-xylidine is classified by the International Agency for Research on Cancer as possibly carcinogenic (group 2B), o-toluidine is classified as carcinogenic to humans (group 1).6 Recently, oral administration of prilocaine has been shown to result in high adduct levels of o-toluidine, a direct proof of its metabolic activation to a carcinogen.7 This should be another reason to avoid therapeutic use of prilocaine when ever possible.

Therapy-induced carboplatin–DNA adduct levels in human ovarian tumours in relation to assessment of adduct measurement in mouse tissues

Despite an increasing understanding of the molecular mechanisms by which platinum drug DNA adducts interact with cellular processes, the relationship between adduct formation in tumours and clinical response remains unclear. We have determined carboplatin–DNA adduct levels in biopsies removed from ovarian cancer patients following treatment. Reliability of DNA adduct measurements in tissues samples were assessed using experimental animals. Platinum–DNA adduct levels were measured using inductively coupled plasma mass spectrometry (ICP-MS) and plasma drug concentrations determined by atomic absorption spectrometry (AAS). Adduct levels in tissues and plasma pharmacokinetics were determined in Balb/c mice exposed to platinum drugs. Comparisons of adduct levels in tumour and normal tissue were made in nu/nu mice carrying human neuroblastoma xenografts. At 30 min post-cisplatin administration, adduct levels in DNA from kidney and liver were approximately 10- and 6-fold higher than spleen or tumour. By 60 min, levels in liver and kidney, but not spleen or tumour, had fallen considerably. Carboplatin showed high adduct levels only in kidney. Adduct levels in tumour xenografts were comparable to those induced in vitro with similar drug exposures. In clinical samples removed 6 h after drug administration, adduct levels ranged from 1.9 to 4.3 and 0.2 to 3.6 nmol Pt/g DNA for tumour biopsies and peripheral blood mononuclear cells, respectively. No correlation was apparent between these two data sets. The present results demonstrate that reliable measurements of adducts in clinical tumours are feasible. Future results should provide insight into drug resistance.

Altered tissue distribution of 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine-DNA adducts in mice transgenic for human sulfotransferases 1A1 and 1A2

Soluble sulfotransferases (SULTs) generate electrophilically reactive metabolites from numerous food-borne compounds, environmental contaminants and drugs, often resulting in mutagenicity and carcinogenicity. Substrate specificity, regulation and tissue distribution of SULTs show large interspecies differences. In humans, therefore, SULTs may be involved in the induction of cancer in different tissues than in standard animal models. To construct a rodent model taking some species differences into account, we transferred a 68.5 kb human (h) genomic sequence that comprised the transcribed and long flanking regions of SULT1A1 and 1A2 into murine oocytes. This approach resulted in several mouse lines expressing these human genes in a copy number-dependent manner with a tissue distribution similar to that in humans. In previous in vitro studies, we had demonstrated that human SULT1A1 and 1A2 efficiently catalyze the terminal activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a mutagen. The transgenic mice were used to study the hSULT1A1/1A2-mediated activation. Tissue distribution and levels of DNA adducts were determined in hSULT1A1/1A2 transgenic and wild-type mice after an oral dosage of PhIP. Transgenic mice exhibited significantly elevated PhIP-DNA adduct levels compared with the wild-type in liver (13-fold), lung (3.8-fold), colon (2-fold), kidney (1.6-fold) and cecum (1.5-fold). Moreover, among the eight tissues examined, liver was the one with the lowest and highest adduct levels in wild-type and transgenic mice, respectively. Hence, expression of hSULT1A1/1A2 not only enhanced the genotoxicity but also substantially changed the organotropism of PhIP.

Heterocyclic aromatic amines form high levels of DNA adducts in human hepatocytes

Heterocyclic aromatic amines form high levels of DNA adducts in human hepatocytes

Toxic Effects of Methylated Benzo[a]pyrenes in Rat Liver Stem-Like Cells

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

Identification of glucosinolate congeners able to form DNA adducts and to induce mutations upon activation by myrosinase

Juices from Brassicales are mutagenic in Salmonella typhimurium and characteristic adducts are formed with the endogenous DNA in Brassicales homogenates. These effects require myrosinase activity, suggesting an involvement of breakdown products of glucosinolates (GLs). We aimed to identify GLs congeners producing these effects. We investigated twelve individual GLs for mutagenicity in S. typhimurium TA104 and TA100 and for adduct formation with herring sperm DNA using the 32P-postlabelling/thin-layer chromatography method. All bacteriotoxic and mutagenic effects observed required the presence of myrosinase. Neoglucobrassicin, 4-methoxyglucobrassicin and sinalbin showed mutagenicity over wide concentration ranges, with neoglucobrassicin being the most potent congener. Six other GLs led to modest increases in the number of revertants in a small concentration range, before toxicity overshadowed this effect. The remaining three GLs showed some toxicity, but no mutagenicity. However, all twelve GLs formed DNA adducts. Clearly the highest adduct levels were detected with the indole GLs tested. They matched the major adduct spots formed in Brassicales homogenates. The observation that GLs are genotoxic demands follow-up studies on possible genotoxic and carcinogenic effects of these common food compounds in animal models and humans. Our study may be used to prioritize the congeners in further studies.

Abstract 3461: DNA adducts of ortho-toluidine in human bladder

Abstract Recently, ortho-toluidine (o-Tol) has been added to the list of aromatic amines in group 1 of human carcinogens by the International Agency for Research on Cancer. High levels of o-Tol hemoglobin adducts have been found in workers of a chemical factory with high excess of bladder cancer (Ward et al., JNCI 88:1046-52, 1996) as well as in patients treated with the local anesthetic prilocaine (Gaber et al., Toxicology 229:157-64, 2007). In contrast to 4-aminobiphenyl (4-ABP), the presence of DNA adducts of o-Tol has not yet been proven in humans. For the present study we obtained bladder tissue from 46 sudden death victims (SDV) and 12 samples from human bladder tumors. DNA was isolated from the epithelial and a submucosal layer of the SDV tissue samples and from tumor samples. For analysis of DNA adducts releasing o-Tol and 4-ABP, the aromatic amines were hydrolyzed from DNA by addition of 100 μL 4 N HCl. As internal standards d9-o-Tol and d5-4-ABP were added prior to hydrolysis. The acidic DNA solution was washed with dichloromethane and the amines were extracted with hexane after alkalinization. Determination of o-Tol and 4-ABP was achieved after derivatization with heptafluorobutyric anhydride by capillary gas chromatography/mass spectrometry with negative chemical ionization. Water samples were analyzed with each batch of samples to control for background contamination. Values less than 2-fold higher than background values were designated as not detectable and included as zero values in calculation of mean±standard error. Adducts of o-Tol well above background levels could be detected in 11 of 12 tumor samples (8.72±1.30 fmol/µg DNA). In contrast, only 4 of 12 tumor samples were positive for ABP (0.057±0.036 fmol/µg; p&amp;lt;0.0001 vs o-Tol). In 46 samples of bladder tissue from SDV, o-Tol DNA adduct levels were significantly lower compared to tumor samples but still detectable in 13 samples from the epithelial (0.24±0.09 fmol/µg DNA; p&amp;lt;0.0001) and 10 samples from the submucosal layer (0.27±0.10 fmol/µg DNA; p&amp;lt;0.0001), respectively. 4-ABP was detectable at significantly lower levels compared to tumor samples in 32 samples from the epithelial (0.011±0.003 fmol/µg DNA; p&amp;lt;0.0001) and 28 samples from the submucosal layer (0.019±0.007 fmol/µg DNA; p&amp;lt;0.005) of the 46 samples of SDV bladder tissue. Further characterization of the data concerning active smoking and polymorphism of N-acetyltransferase 2 genotype is underway. In conclusion, for the first time DNA adducts releasing o-Tol were detected in human urinary bladder. The presence of DNA adducts in the human target tissue of o-Tol strongly support a carcinogenic risk which should be taken into account in preventive hazard minimization in both occupational medicine and drug therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3461.

Lifestyle, Environmental, and Genetic Predictors of Bulky DNA Adducts in a Study Population Nested within a Prospective Danish Cohort

Bulky DNA adducts are considered a potential biomarker of cancer risk. In this study, the association between various lifestyle, environmental, and genetic factors and the levels of bulky DNA adducts in peripheral leukocytes was examined in a study group nested within a population-based prospective Danish cohort. At enrollment, blood samples were collected and information on lifestyle, including dietary and smoking habits, obtained. Previously, bulky DNA adducts were measured in 245 individuals who developed lung cancer and 255 control members of the cohort. Of these 500 individuals, data on 375 individuals were included in this study, excluding 125 cases, which developed lung cancer within the first 3 yr after blood sampling. Bulky DNA adduct levels were measured by 32 P-postlabeling technique and polymorphisms in carcinogen metabolism and DNA repair genes were determined. Potential predictors of bulky DNA adduct levels were analyzed by univariate and multivariate regression analyses. Women tended to have higher adduct levels than men. Living in central Copenhagen and surface darkness of fried meat and fish were associated with quantitative higher adduct levels. No significant associations were found between dietary factors or smoking and DNA adduct levels. Further, the results showed no prominent associations between any of 12 genetic polymorphisms and adduct levels. Overall, our study showed only few associations between dietary, environmental, and genetic factors and levels of bulky DNA adducts measured in peripheral leukocytes in a general Danish population.

Physical activity and lung cancer among non-smokers: a pilot molecular epidemiological study within EPIC

The association between physical activity, potential intermediate biomarkers and lung cancer risk was investigated in a study of 230 cases and 648 controls nested within the European Prospective Investigation of Cancer and Nutrition. Data on white blood cell aromatic-DNA adducts by 32P-post-labelling and glutathione (GSH) in red blood cells were available from a subset of cases and controls. Compared with the first quartile, the fourth quartile of recreational physical activity was associated with a lower lung cancer risk (odds ratio (OR) 0.56, 95% confidence interval (CI) 0.35–0.90), higher GSH levels (+1.87 μmol GSH g−1 haemoglobin, p = 0.04) but not with the presence of high levels of adducts (OR 1.05, 95% CI 0.38–2.86). Despite being associated with recreational physical activity, in these small-scale pilot analyses GSH levels were not associated with lung cancer risk (OR 0.95, 95% CI 0.84–1.07 per unit increase in GSH levels). Household and occupational activity was not associated with lung cancer risk or biomarker levels.

Depurinating Estrogen–DNA Adducts in the Etiology and Prevention of Breast and Other Human Cancers

Experiments on estrogen metabolism, formation of DNA adducts, mutagenicity, cell transformation and carcinogenicity have led to and supported the hypothesis that the reaction of specific estrogen metabolites, mostly the electrophilic catechol estrogen-3,4-quinones, with DNA can generate the critical mutations to initiate breast and other human cancers. Analysis of depurinating estrogen-DNA adducts in urine demonstrates that women at high risk of, or with breast cancer, have high levels of the adducts, indicating a critical role for adduct formation in breast cancer initiation. Men with prostate cancer or non-Hodgkin lymphoma also have high levels of estrogen-DNA adducts. This knowledge of the first step in cancer initiation suggests the use of specific antioxidants that can block formation of the adducts by chemical and biochemical mechanisms. Two antioxidants, N-acetylcysteine and resveratrol, are prime candidates to prevent breast and other human cancers because in various M in vitro and in vivo experiments, they reduce the formation of estrogen-DNA adducts.

Malondialdehyde–Deoxyguanosine Adducts among Workers of a Thai Industrial Estate and Nearby Residents

BackgroundHumans living near industrial point emissions can experience high levels of exposures to air pollutants. Map Ta Phut Industrial Estate in Thailand is the location of the largest steel, oil refinery, and petrochemical factory complexes in Southeast Asia. Air pollution is an important source of oxidative stress and reactive oxygen species, which interact with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively.ObjectiveWe measured the levels of malondialdehyde–deoxyguanosine (dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in petrochemical workers, nearby residents, and subjects living in a control district without proximity to industrial sources.DesignWe conducted a cross-sectional study to compare the prevalence of malondialdehyde-dG adducts in groups of subjects experiencing various degrees of air pollution.ResultsThe multivariate regression analysis shows that the adduct levels were associated with occupational and environmental exposures to air pollution. The highest adduct level was observed in the steel factory workers. In addition, the formation of DNA damage tended to be associated with tobacco smoking, but without reaching statistical significance. A nonsignificant increase in DNA adducts was observed after 4–6 years of employment among the petrochemical complexes.ConclusionsAir pollution emitted from the Map Ta Phut Industrial Estate complexes was associated with increased adduct levels in petrochemical workers and nearby residents. Considering the mutagenic potential of DNA lesions in the carcinogenic process, we recommend measures aimed at reducing the levels of air pollution.

Aromatic DNA adducts in relation to dietary and other lifestyle factors in Spanish adults

The aim of this study was to assess the relationship between bulky DNA adducts in white blood cells (WBC) and lifestyle factors in a sample from the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC). 296 subjects aged between 35 and 64 years, from five regions, were included. Food intake was estimated with a computerized version of dietary history questionnaire. Daily intake of polycyclic aromatic hydrocarbons (PAH) was estimated using a database with information on food content of potential carcinogens. Data on lifestyle and health factors were collected and DNA adducts measured using the nuclease P1 32P-postlabelling technique. Geometric means of adducts were similar for men and women (4.11/109 and 3.94/109 nucleotides, respectively). Highest levels of adduct were observed in non-smokers and non-occupationally exposed. Meat intake, oils and fats were associated with higher levels of adducts, but all non-statistically significant. Higher intakes of calcium, sodium and phosphorus were associated with lower adducts levels. Summarising, our study shows that bulky adducts measured by 32P-postlabelling in DNA from WBC do not correlate with the usual diet of healthy Spanish adults. Although it has been proposed that diet be the main source of PAH in nonsmokers without occupational exposure, DNA adducts do not seem to be suitable biomarkers of dietary PAH in general population.

Toward an “omic” physiopathology of reactive chemicals: Thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds

Cancer and degenerative diseases are major causes of morbidity and death, derived from the permanent modification of key biopolymers such as DNA and regulatory proteins by usually smaller, reactive molecules, present in the environment or generated from endogenous and xenobiotic components by the body's own biochemical mechanisms (molecular adducts). In particular, protein adducts with organic electrophiles have been studied for more than 30 [see, e.g., Calleman et al., 1978] years essentially for three purposes: (a) as passive monitors of the mean level of individual exposure to specific chemicals, either endogenously present in the human body or to which the subject is exposed through food or environmental contamination; (b) as quantitative indicators of the mean extent of the individual metabolic processing which converts a non-reactive chemical substance into its toxic products able to damage DNA (en route to cancer induction through genotoxic mechanisms) or key proteins (as in the case of several drugs, pesticides or otherwise biologically active substances); (c) to relate the extent of protein modification to that of biological function impairment (such as enzyme inhibition) finally causing the specific health damage. This review describes the role that contemporary mass spectrometry-based approaches employed in the qualitative and quantitative study of protein-electrophile adducts play in the discovery of the (bio)chemical mechanisms of toxic substances and highlights the future directions of research in this field. A particular emphasis is given to the measurement of often high levels of the protein adducts of several industrial and environmental pollutants in unexposed human populations, a phenomenon which highlights the possibility that a number of small organic molecules are generated in the human organism through minor metabolic processes, the imbalance of which may be the cause of "spontaneous" cases of cancer and of other degenerative diseases of still uncharacterized etiology. With all this in mind, it is foreseen that a holistic description of cellular functions will take advantage of new analytical methods based on time-integrated metabolomic measurements of a new biological compartment, the "adductome," aimed at better understanding integrated organism response to environmental and endogenous stressors.