Diol Epoxide Research Articles

Interaction of benzo[a]pyrene with other risk factors in hepatocellular carcinoma: a case-control study in Xiamen, China

PurposeLarge epidemiologic studies about the relationship between benzo[a]pyrene (B[a]P) and hepatocellular carcinoma (HCC) have been limited. B[a]P diol epoxide (BPDE) is a highly reactive metabolite of B[a]P that binds covalently to form DNA adducts. We evaluated the interaction between B[a]P exposure with other risk factors in HCC, in a case-control study of 345 HCC and 961 healthy controls. MethodsConcentration of BPDE-DNA adducts in blood was determined by enzyme-linked immunosorbent assay. The interaction between BPDE-DNA adducts and other risk factors on HCC were evaluated by multivariate logistic regression analysis. ResultsMean concentration of BPDE-DNA adducts in blood of cases was significantly higher than that of the controls. The risk of HCC increased with elevated concentration of BPDE-DNA adducts (x2 = 203.57, Ptrend < .001) and the odds ratio was 7.44 (95% confidence interval, 5.29–10.45) for the first versus fourth quartile of adduct levels. The relative excess risk due to interaction between BPDE-DNA adducts and hepatitis B virus surface antigen and drinking was 34.71 and 54.92, and the attributable proportion due to interaction was 41.53% and 75.59%, respectively. ConclusionsThe high level of BPDE-DNA adducts in blood is associated with HCC and that environmental exposure to B[a]P may increase the risk of HCC, especially among drinkers and populations with hepatitis B virus infection.

HspA1A facilitates DNA repair in human bronchial epithelial cells exposed to Benzo[a]pyrene and interacts with casein kinase 2

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant that induces deoxyribonucleic acid (DNA) damage. The inducible heat shock protein (HspA1A) can function as a molecular chaperone; however, its role in DNA repair remains largely unknown. In the present study, human bronchial epithelial cells (16HBE) stably transfected with plasmids carrying HspA1A gene or shRNAs against HspA1A were treated with BaP. DNA damage levels of the cells were evaluated by comet assay. Results suggest that HspA1A could protect cells against DNA damage and facilitate the decrease of DNA damage levels during the first 2h of DNA repair. DNA repair capacity (DRC) of Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts was evaluated by host cell reactivation assay in the stable 16HBE cells transfected with luciferase reporter vector PCMVluc pretreated with BPDE. Compared with control cells, cells overexpressing HspA1A showed higher DRC (p < 0.01 at 10μM BPDE and p < 0.05 at 20μM BPDE, respectively), while knockdown of HspA1A inhibited DNA repair (p < 0.05 at 10μM BPDE). Moreover, casein kinase 2 (CK2) was shown to interact with HspA1A by mass spectrometry and co-immunoprecipitation assays. The two proteins were co-localized in the cell nucleus and perinuclear region during DNA repair, and were identified by confocal laser scanning microscope. In addition, cells overexpressing HspA1A showed an increased CK2 activity after BaP treatment compared with control cells (p < 0.01). Our results suggest that HspA1A facilitates DNA repair after BaP treatment. HspA1A also interacts with CK2 and enhances the kinase activities of CK2 during DNA repair.

Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) is mediated mainly by cytochrome P450 monooxygenases (CYP) CYP1A1, 1A2 and 1B1. Several PAH are known to induce these CYP via aryl hydrocarbon receptor (AhR) signaling. Recently, it was shown that the PAH benzo[a]pyrene (BaP) can induce CYP3A4 as well. The induction was suggested to be mediated by the pregnane X receptor (PXR) rather than AhR. Metabolism by CYP3A4 is only known for dihydrodiol metabolites of PAH but not for their parent compounds.In the present study, a CYP3A4 reporter gene assay, requiring the overexpression of PXR, was used to investigate whether the PAH parent compounds BaP, benzo[c]phenanthrene (BcP) and dibenzo[a,l]pyrene (DBalP) as well as their corresponding phase I metabolites, the respective dihydrodiols and diol epoxides, can induce CYP3A4 promoter activity. BaP, BcP and their dihydrodiols were found to significantly activate the CYP3A4 promoter. Moreover, activation of PXR by all four compounds was detected by using a PXR transactivation assay, supporting that PXR mediates CYP3A4 induction by PAH. Taken together, these results show that both PAH parent compounds as well as their phase I metabolites induce CYP3A4 promoter via the transcription factor PXR.

P1.034 Biomarkers of Cigarette Smoking and Association with the Vaginal Microbiota

BackgroundSmoking has been identified in observational studies as a risk factor for bacterial vaginosis (BV). The anti-estrogenic effect of smoking and trace amounts of benzo[a]pyrene diol epoxide (BPDE) may predispose...

Effect of blood storage conditions on DNA repair capacity measurements in peripheral blood mononuclear cells

Due to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken.We performed a genotype–phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p<0.0001) and as benzo[a]pyrene diol epoxide (BPDE)-induced damage repair by the comet assay (p=0.05). No apparent effect of the blood storage conditions on the outcome of γ-ray induced H2AX phosphorylation assay was reported.These results prompted us to further analyze the effects of blood storage conditions by performing a validation study. Three blood samples were simultaneously taken from ten healthy donors, PBMCs were isolated and cryopreserved as follows: immediately after blood collection (day 1); on the following day, after blood storage at RT (day 2-RT); or after blood storage at 4°C (day 2-4°C). DRC was then evaluated using phenotypic assays. The γ-ray induced H2AX phosphorylation assay has been confirmed as the only assay that showed good reproducibility independently of the blood storage conditions. The measurement of OGG assay was most affected by the blood storage conditions.

Mutagenicity and tumorigenicity of the four enantiopure bay-region 3,4-diol-1,2-epoxide isomers of dibenz[a,h]anthracene.

Each enantiomer of the diastereomeric pair of bay-region dibenz[a,h]anthracene 3,4-diol-1,2-epoxides in which the benzylic 4-hydroxyl group and epoxide oxygen are either cis (isomer 1) or trans (isomer 2) were evaluated for mutagenic activity. In strains TA 98 and TA 100 of Salmonella typhimurium, the diol epoxide with (1S,2R,3S,4R) absolute configuration [(-)-diol epoxide-1] had the highest mutagenic activity. In Chinese hamster V-79 cells, the diol epoxide with (1R,2S,3S,4R) absolute configuration [(+)-diol epoxide-2] had the highest mutagenic activity. The (1R,2S,3R,4S) diol epoxide [(+)-diol epoxide-1] also had appreciable activity, whereas the other two bay-region diol epoxide enantiomers had very low activity. In tumor studies, the (1R,2S,3S,4R) enantiomer was the only diol epoxide isomer tested that had strong activity as a tumor initiator on mouse skin and in causing lung and liver tumors when injected into newborn mice. This stereoisomer was about one-third as active as the parent hydrocarbon, dibenz[a,h]anthracene as a tumor initiator on mouse skin; it was several-fold more active than dibenz[a,h]anthracene as a lung and liver carcinogen when injected into newborn mice. (-)-(3R,4R)-3β,4α-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(-)-3,4-dihydrodiol] was slightly more active than dibenz[a,h]anthracene as a tumor initiator on mouse skin, whereas (+)-(3S,4S)-3α,4β-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(+)-3,4-dihydrodiol] had only very weak activity. The present investigation and previous studies with the corresponding four possible enantiopure bay-region diol epoxide enantiomers/diastereomers of benzo[a]pyrene, benz[a]anthracene, chrysene, benzo[c]phenanthrene, dibenz[c,h]acridine, dibenz[a,h]acridine and dibenz[a,h]anthracene indicate that the bay-region diol epoxide enantiomer with [R,S,S,R] absolute stereochemistry has high tumorigenic activity on mouse skin and in newborn mice.

RIP1 potentiates BPDE-induced transformation in human bronchial epithelial cells through catalase-mediated suppression of excessive reactive oxygen species

Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer and mouse lung tumor tissues. RIP1 expression was remarkably increased in cigarette smoke-exposed mouse lung. In human bronchial epithelial cells (HBECs), RIP1 was significantly induced by cigarette smoke extract or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo(a)pyrene. In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs), including c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The ROS-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced ROS accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production.

Adenine–DNA Adducts Derived from the Highly Tumorigenic Dibenzo[a,l]pyrene Are Resistant to Nucleotide Excision Repair while Guanine Adducts Are Not

The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N(2)-dG) and adenine (N(6)-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N(2)-dG linkage site is ∼35 times more susceptible to NER dual incisions than the stereochemically identical N(6)-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar but somewhat smaller effect (factor of ∼15) is observed. The striking resistance of the bulky N(6)-dA in contrast to the modest to good susceptibilities of the N(2)-dG adducts to NER is interpreted in terms of the balance between lesion-induced DNA distorting and DNA stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA.

Mechanisms of oral carcinogenesis induced by dibenzo[a,l]pyrene: An environmental pollutant and a tobacco smoke constituent

We previously reported that dibenzo[a,l]pyrene (DB[a,l]P), the most potent known environmental carcinogen among polycyclic aromatic hydrocarbons (PAH) congeners, is carcinogenic in the oral tissues of mice. We have now developed a new mouse model which employs the oral application of the fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE), a metabolite of the tobacco smoke constituent DB[a,l]P, and we show its specific induction of oral squamous cell carcinoma (OSCC) in both tongue and other oral tissues. Groups of B6C3F1 mice (20/group) received 6 or 3 nmol of (±)-anti-DB[a,l]PDE administered into the oral cavity; 3 times per week for 38 weeks. Additional groups received the vehicle alone or were left untreated. Mice were sacrificed 42 weeks after the first carcinogen administration. The high dose induced 74 and 100% OSCC in the tongue and other oral tissues, respectively; the corresponding values at the lower dose were 45 and 89%. Using immunohistochemistry, we showed that DB[a,l]PDE resulted in overexpression of p53 and COX-2 proteins in malignant tissues when compared to normal oral tissues and tongues. Consistent with the carcinogenicity, we demonstrated powerful mutagenicity in cII gene in B6C3F1 (Big Blue) mouse tongue. The mutational profile in lacI reporter gene is similar to those detected in human head and neck cancer, and p53 mutations were observed in mouse oral tumor tissues. Taken together, we conclude that the formation of diol epoxides plays a major role among the mechanisms by which DB[a,l]P exerts its oral mutagenicity and tumorigenicity.

Kinetics of naphthalene metabolism in target and non-target tissues of rodents and in nasal and airway microsomes from the Rhesus monkey

Naphthalene produces species and cell selective injury to respiratory tract epithelial cells of rodents. In these studies we determined the apparent Km, Vmax, and catalytic efficiency (Vmax/Km) for naphthalene metabolism in microsomal preparations from subcompartments of the respiratory tract of rodents and non-human primates. In tissues with high substrate turnover, major metabolites were derived directly from naphthalene oxide with smaller amounts from conjugates of diol epoxide, diepoxide, and 1,2- and 1,4-naphthoquinones. In some tissues, different enzymes with dissimilar Km and Vmax appeared to metabolize naphthalene. The rank order of Vmax (rat olfactory epithelium>mouse olfactory epithelium>murine airways≫rat airways) correlated well with tissue susceptibility to naphthalene. The Vmax in monkey alveolar subcompartment was 2% that in rat nasal olfactory epithelium. Rates of metabolism in nasal compartments of the monkey were low. The catalytic efficiencies of microsomes from known susceptible tissues/subcompartments are 10 and 250 fold higher than in rat airway and monkey alveolar subcompartments, respectively. Although the strong correlations between catalytic efficiencies and tissue susceptibility suggest that non-human primate tissues are unlikely to generate metabolites at a rate sufficient to produce cellular injury, other studies showing high levels of formation of protein adducts support the need for additional studies.

Abstract 3586: Benzopyrene diol epoxide attenuates p34cdc2 and inhibits cell growth: role of p53.

Abstract Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers cellular protective response of cell growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblast associated with accumulation of p53 protein, a known cell growth inhibitory transcription factor. Here we report that in different cell lines BPDE treatment (ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) triggers variable extent of cell growth inhibition which do not correspond to the extent of increased p53 accumulation as we observed. We also observed that BPDE treatment of cells significantly down-regulates expression of p34cdc2, a known cell cycle activating protein. Although the role of cdc2 down-regulation in inhibition of cell cycle progression is well known, to the best of our knowledge cdc2 down-regulation in response to cellular insult by PAHs has not been reported. Unlike p53 accumulation there is correspondence between extent of cell growth inhibition and the extent of cdc2 down-regulation by BPDE in different cell lines. Interestingly, BPDE-induced cdc2 down-regulation is observed to be p53 dependent although there is lack of correspondence between the extent of p53 accumulation and cdc2 down-regulation. However, extent of BPDE induced cdc2 down-regulation was observed to have correspondence with the extent of accumulation of cell cycle inhibitor protein p21 (transactivation product of p53) in different cell lines. These findings may have an implication that cell growth inhibition in response to DNA damaging PAHs may involve down-regulation of cdc2 protein mediated by p53 activation (transactivation ability), and the extent of p53 accumulation is not the determining factor in this regard. Citation Format: Jagat J. Mukherjee, Subodh Kumar. Benzopyrene diol epoxide attenuates p34cdc2 and inhibits cell growth: role of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3586. doi:10.1158/1538-7445.AM2013-3586

Abstract 3602: Effect of alcohol on DB[a,l]P, an environmental pollutant and a tobacco smoke constituent, on DNA adduct formation in mouse oral tissues.

Abstract Alcohol acts synergistically with tobacco smoke to greatly increase the risk of oral squamous cell carcinoma (OSCC) up to 20-fold; the mechanisms of this synergism remain unknown. Research in this area has been hampered by the lack of appropriate animal models that would mimic chronic simultaneous human exposure to alcohol and representative carcinogens in tobacco or tobacco smoke as a whole. Recently, we developed a more relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. We also showed that its diol epoxide metabolite, (±)-anti-DB[a,l]PDE, is a potent and specific carcinogen in tongue and other oral tissues in mice. We hypothesize that chronic alcohol consumption will increase the carcinogenic potency of DB[a,l]P in the mouse oral cavity by multiple mechanisms including the depletion of glutathione (GSH) and increasing levels of covalent DB[a,l]PDE-DNA adducts. As an initial study to test our hypothesis, mice were fed Isocaloric Lieber-DeCarli-type liquid diets formulated by BioServ (Frenchtown, N.J.) with or without ethanol (35% of total calories, increased gradually from 0, 11, 22 to 35%). After 3 weeks, mice were treated with 2 daily doses of DB[a,l]P (240 nmol each) and/or tobacco smoke particulates (TSP, 2R4F, 2.5 mg/mouse). Mice were sacrificed 24 hrs later and DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We showed that alcohol consumption significantly increased the levels of (-)-anti-trans-DB[a,l]P-dA in the absence of TSP (25%) or in the presence of TSP (75%). We also demonstrated that GSH levels in buccal mucosa were decreased (38%) in mice fed with liquid diet (35% calories from ethanol) compared with mice fed with control diet (0.89±0.13 μmol/g vs 1.23±0.17 μmol/g). Collectively, our preliminary results support our hypothesis that alcohol consumption will increase the carcinogenic potency of DB[a,l]P by altering the metabolic detoxification via depleting GSH and increasing DNA damage in oral tissues of mice. Citation Format: Shang-Min Zhang, Kun-Ming Chen, John R. Richie, Ana Calcagnotto, Cesar Aliaga, Arthur Berg, Yuan-Wan Sun, Arun K. Sharma, Shantu Amin, Karam El-Bayoumy. Effect of alcohol on DB[a,l]P, an environmental pollutant and a tobacco smoke constituent, on DNA adduct formation in mouse oral tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2013-3602

Abstract 2910: Suppressing excessive reactive oxygen species accumulation by RIP1 potentiates BPDE-induced transformation of human bronchial epithelial cells.

Abstract Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer (NSCLC) tissues and cell lines. RIP1 expression in human bronchial epithelial cells (HBECs) was significantly induced by cigarette smoke extract (CSE) or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo (a) pyrene (BaP). In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS), mainly hydrogen peroxide (H2O2), and activation of mitogen-activated protein kinases (MAPKs) including JNK, ERK and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The H2O2-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced H2O2 accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production. Citation Format: Qiong Wang, Wenshu Chen, Xiuling Xu, Bilan Li, Weiyang He, Mabel T. Padilla. Suppressing excessive reactive oxygen species accumulation by RIP1 potentiates BPDE-induced transformation of human bronchial epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2910. doi:10.1158/1538-7445.AM2013-2910

Longitudinal study of [D10]phenanthrene metabolism by the diol epoxide pathway in smokers

The extent of metabolism of [D10]phenanthrene to [D10]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetradeuterophenanthrene ([D10]PheT) could be a biomarker of human metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, leading to identification of smokers particularly susceptible to lung cancer. The longitudinal stability of [D10]PheT was evaluated in 24 cigaret smokers given 7–8 oral doses of [D10]phenanthrene (10 µg) over 5.5 months. [D10]PheT in 6 h urine was quantified after each dose. The overall coefficient of variation for 24 subjects was (mean ± S.D.) 27.4% ± 8.83%. Thus, a single administration of [D10]phenanthrene is likely sufficient to determine a smoker’s ability to metabolize it to [D10]PheT.

A sandwich ELISA for measuring benzo[a]pyrene–albumin adducts in human plasma

Exposure to polycyclic aromatic hydrocarbons has often been quantified via DNA or human serum albumin (HSA) adducts of the carcinogenic metabolite benzo[a]pyrene diol epoxide (BPDE). We previously reported a sandwich ELISA, using 8E11 as capture antibody and anti-HSA as detection antibody, that detected intact BPDE adducts in HSA isolated from plasma. After confirming that BPDE binds to HSA at His146 and Lys195, we modified the ELISA to measure intact BPDE–HSA directly in human plasma. To adjust for interference due to nonspecifically bound HSA on well surfaces and to cross-reactivity of the antibodies, the ELISA employs paired wells with and without addition of BPDE tetrols to deactivate 8E11. By performing assays in quadruplicate, a series of sample-specific adjustments and screening steps are used to reduce measurement errors that are a consequence of detecting low BPDE–HSA concentrations in the general population. ELISA measurements of BPDE–HSA in plasma from smoking and nonsmoking subjects (range 0.280–2.88 ng BPDE–HSA/mg HSA) and from highway workers with and without exposure to asphalt emissions (range 0.346–13.9 ng BPDE–HSA/mg HSA) detected differences in BPDE–HSA levels in the a priori expected directions.

Plastic antibody for DNA damage: fluorescent imaging of BPDE–dG adducts in genomic DNA

Exogenous and endogenous genotoxic carcinogens and their in vivo metabolites may result in DNA damage and cause adverse health effects. It is very difficult to specifically detect trace DNA damage in the presence of a large excess of unmodified nucleosides. Here we report a molecularly imprinted polymer (MIP) nanocomposite, namely nanoMIP, which can be used as a "plastic antibody" for specific recognition of benzo[a]pyrene diol epoxide (BPDE)-DNA adduct. Enhanced binding affinity (880 nM) of nanoMIPs was achieved by using two tailor-made functional monomers. The property of binding kinetics was greatly improved in virtue of the well-defined nanostructure, which was fabricated by initiators for continuous activator regeneration-atom transfer radical polymerization (ICAR-ATRP). The BPDE-adducted DNA can be specifically captured by synthetic nanoMIP. By taking advantage of this antibody-mimicking behavior, we further developed a fluorescently imaged particle counting immunoassay (FIPCIA) method for ultrasensitive detection of BPDE-ssDNA adducts using a laser scanning confocal microscope (LSCM). The number of countable fluorescent dots is proportional to the content of BPDE-ssDNA adducts in the DNA sample. The proposed plastic antibody-based FIPCIA method can detect traces of BPDE-DNA adducts as low as 18 pM in human lung carcinoma A549 cells. This highly-sensitive detection of DNA lesions offers a promising alternative to immunogenic antibody-based immunoassays for genomics and DNA modification analysis.

Further investigations on the modified comet assay for measuring aphidicolin-block nucleotide excision repair

The comet assay is increasingly used to measure the repair of various types of DNA damage. Modifications of the standard protocol have been introduced to determine the repair capacity of specific DNA repair pathways by the removal of pathway-specific DNA lesions. Recently, a cellular phenotype assay for nucleotide excision repair (NER) by quantifying the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells with benzo[a]pyrene diol epoxide (BPDE) in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC) was developed (Vande Loock, K., Decordier, I., Ciardelli, R., Haumont, D. and Kirsch-Volders, M. (2010) An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity. Mutagenesis, 25, 25-32). Individual repair capacity (RC) was defined as the amount of DNA damage induced by BPDE in the presence of APC minus the damage induced by BPDE and APC alone. This value should mainly reflect the incision capacity of the NER enzymes. Following this approach, we investigated the RC of cultured isolated peripheral blood mononuclear cells of nine donors in repeated experiments. We also performed the same experiments with peripheral whole blood cultures from these donors. Our results indicated considerable intra- and inter-individual variability and substantial differences between the RC of isolated mononuclear cells and whole blood from the same donor. Furthermore, the RC of unstimulated blood did not differ significantly from the repair capacity of stimulated blood but also showed considerable inter-individual variability. Altogether, our results suggest that there is still need for standardisation and validation of this assay before it can be reliably used in human biomonitoring studies.

Study of the Relation Between the Theoretical Descriptors Derived From Ab-initio Calculations with the Carcinogenity of Some Poly Aromatic Hydrocarbons

The various types of descriptors such as ionization energy, molecular hardness, electrophilicity, frontier molecular orbital energies together with electron densities of each atom for the optimized geometries of the molecule of different polyaromatic hydrocarbons (PAHs) were estimated by employing on ab-initio method expressed by Hartree – Fock (HF) model performed at the (3-21G) level of theory. After that these theoretical parametrs were related to the activity carcinogenity of these compounds as carcinogenic agents. The results showed that two factors can be related to carcinogenity of these compounds; the first one is the carbon atoms of low electron densities (i.e the position of electrophilic atoms), the second is the hardness of these compounds. We have also used electron density to highlight the possible strengths of interactions of PAHs with DNA of living cells. On the bases that the main metabolic pathway for activation of these compounds involves formation of bay-region diol epoxide, then, the benzylic carbocations generated from these electrophilic diol epoxide by opening of the epoxide ring are capable of forming covalent adducts with the nucleophilic site in DNA which represent the main factors of carcinogenity of these compounds since adduct is accepted as a critical step in the mechanism by which (PAHs) can cause a genetic mutation resulting inductions of cancer.

Nuclear Magnetic Resonance Solution Structure of an N2-Guanine DNA Adduct Derived from the Potent Tumorigen Dibenzo[a,l]pyrene: Intercalation from the Minor Groove with Ruptured Watson–Crick Base Pairing

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

Abstract A69: Pilot study of the effects of indole-3-carbinol on deuterated phenanthrene metabolism in smokers

Abstract Background/Purpose: Polycyclic aromatic hydrocarbons (PAH), widely considered as critical causative agents of tobacco-related lung cancer, are found in high levels in tobacco smoke and require metabolic activation to exert their carcinogenic effects. The development of urinary biomarkers of PAH metabolism using the more abundant and generally non-carcinogenic phenanthrene (Phe) as a surrogate of the prototypic carcinogenic PAH benzo[a]pyrene (BaP) has provided a reliable tool to characterize the uptake and metabolism of BaP in smokers. There is wide inter-individual variation in PheT metabolism. Furthermore, smokers who are more efficient metabolic activators of Phe appear to have a higher risk of developing lung cancer in prospective case-control studies. We examined the effects of indole-3-carbinol (I3C), a compound arising from cruciferous vegetables shown to possess anti-cancer activity, in modifying Phe metabolism. We hypothesized that I3C might have a chemopreventive effect in cigarette smokers by abrogating the metabolic activation and/or detoxification of [D10]Phe. Procedures: Ten micrograms of [D10]Phe was administered via inhalation of a smoked cigarette prior to and after 7 days if I3C 400 mg po BID. Plasma and urine samples were collected at baseline and for 48 hours following [D10]Phe administration. Deuterated phenanthrene tetraol ([D10]PheT, a reflection of uptake and metabolic activation via the diol epoxide pathway), and deuterated phenanthrol ([D10]HOPhe, a reflection of detoxification and elimination) were quantified in urine and plasma before and after I3C and compared. Results: A total of 8 subjects completed the study procedures, 6 females and 2 males. Subject 8 was clearly an outlier and was excluded from analysis. One-sample t-test was used to compare the mean difference in [D10]PheT and the mean difference in [D10]HOPhe among the group before and after I3C. The reduction in [D10]PheT in urine was significant (one-sample t-test, p=.023), although only marginally so in plasma (=.083). [D10]PheT AUC was not reduced (p=.23). [D10]HOPhe was generally reduced but not significantly (p=.15). Correlation between 6-hour and 48-hour [D10]PheT and [D10]HOPhe was very strong (r=.873; p=.010 and r=.968;p&amp;lt;.001, respectively). Conclusion: I3C may be an effective chemopreventive agent in tobacco-related lung cancer by reducing the metabolic activation of carcinogenic PAH. A larger, definitive study is needed to confirm these results. Citation Format: Naomi Fujioka, Yan Zhong, J. Bradley Holchalter, Diane Rauch, Joni J. Jensen, Chap T. Le, Dorothy K. Hatsukami, Stephen S. Hecht. Pilot study of the effects of indole-3-carbinol on deuterated phenanthrene metabolism in smokers. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A69.