Benzo[a]pyrene Diol Epoxide Research Articles

Dysfunction of methionine sulfoxide reductases to repair damaged proteins by nickel nanoparticles

BackgroundProtein oxidation is considered to be one of the main causes of cell death, and methionine is one of the primary targets of reactive oxygen species (ROS). However, the mechanisms by which nickel nanoparticles (NiNPs) cause oxidative damage to proteins remain unclear. ObjectivesThe objective of this study is to investigate the effects of NiNPs on the methionine sulfoxide reductases (MSR) protein repairing system. MethodsTwo physically similar nickel-based nanoparticles, NiNPs and carbon-coated NiNP (C-NiNPs; control particles), were exposed to human epithelial A549 cells. Cell viability, benzo(a)pyrene diolepoxide (BPDE) protein adducts, methionine oxidation, MSRA and B3, microtubule-associated protein 1A/1B-light chain 3 (LC3) and extracellular signal-regulated kinase (ERK) phosphorylation were investigated. ResultsExposure to NiNPs led to a dose-dependent reduction in cell viability and increased BPDE protein adduct production and methionine oxidation. The methionine repairing enzymatic MSRA and MSRB3 production were suppressed in response to NiNP exposure, suggesting the oxidation of methionine to MetO by NiNP was not reversed back to methionine. Additionally, LC3, an autophagy marker, was down-regulated by NiNPs. Both NiNP and C-NiNP caused ERK phosphorylation. LC3 was positively correlated with MSRA (r=0.929, p<0.05) and MSRB3 (r=0.893, p<0.05). ConclusionsMSR was made aberrant by NiNP, which could lead to the dysfunction of autophagy and ERK phosphorylation. The toxicological consequences may be dependent on the chemical characteristics of the nanoparticles.

TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in (Hupki) mouse embryo fibroblasts

Somatic mutations in the tumour suppressor gene TP53 occur in more than 50% of human tumours; in some instances exposure to environmental carcinogens can be linked to characteristic mutational signatures. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay (HIMA) is a useful model for studying the impact of environmental carcinogens on TP53 mutagenesis. In an effort to increase the frequency of TP53-mutated clones achievable in the HIMA, we generated nucleotide excision repair (NER)-deficient HUFs by crossing the Hupki mouse with an Xpa-knockout (Xpa-Null) mouse. We hypothesized that carcinogen-induced DNA adducts would persist in the TP53 sequence of Xpa-Null HUFs leading to an increased propensity for mismatched base pairing and mutation during replication of adducted DNA. We found that Xpa-Null Hupki mice, and HUFs derived from them, were more sensitive to the environmental carcinogen benzo[a]pyrene (BaP) than their wild-type (Xpa-WT) counterparts. Following treatment with the reactive metabolite of BaP, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), Xpa-WT and Xpa-Null HUF cultures were subjected to the HIMA. A significant increase in TP53 mutations on the transcribed strand was detected in Xpa-Null HUFs compared to Xpa-WT HUFs, but the TP53-mutant frequency overall was not significantly different between the two genotypes. BPDE induced mutations primarily at G:C base pairs, with approximately half occurring at CpG sites, and the predominant mutation type was G:C>T:A in both Xpa-WT and Xpa-Null cells. Further, several of the TP53 mutation hotspots identified in smokers’ lung cancer were mutated by BPDE in HUFs (codons 157, 158, 245, 248, 249, 273). Therefore, the pattern and spectrum of BPDE-induced TP53 mutations in the HIMA are consistent with TP53 mutations detected in lung tumours of smokers. While Xpa-Null HUFs exhibited increased sensitivity to BPDE-induced damage on the transcribed strand, NER-deficiency did not enhance TP53 mutagenesis resulting from damage on the non-transcribed strand in this model.

Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in Saccharomyces cerevisiae.

During Nucleotide Excision Repair (NER) in the yeast S. cerevisiae, ubiquitylation of Rad4 is carried out by the E3 ubiquitin ligase that includes Rad7-Elc1-Cul3 and is critical to optimal NER. Rad7 E3 activity targets Rad4 for degradation by the proteaseome but, in principle, could also trigger other DNA damage responses. We observed increased nuclear ubiquitin foci (Ub-RFP) formation in S. cerevisiae containing a Rad7 E3 ligase mutant (rad7SOCS) in response to DNA damage by benzo[a]pyrenediolepoxide (BPDE) in dividing cells. Immunoblots reveal that ubiquitin conjugates of Rpn10 and Dsk2 accumulate in greater abundance in rad7SOCS compared to RAD7 in dividing cells in response to BPDE which makes Rpn10 and Dsk2 candidates for being the ubiquitylated species observed in our microscopy experiments. Microscopy analysis with strains containing Dsk2-GFP shows that Dsk2-GFP and Dsk2-GFP/Ub-RFP colocalized in nuclear foci form to an increased extent in a rad7SOCS mutant background in dividing cells than in a RAD7 wild-type strain. Further, Dsk2-GFP in the rad7SOCS strain formed more foci at the plasma membrane following BPDE treatment in dividing cells relative to strains containing RAD7 or a rad7Δ deletion mutant. In response to a different agent, UV irradiation, levels of ubiquitylated proteins were increased in rad7SOCS relative to RAD7, and the proteasomal deubiquitylase subunit, Rpn11 was even monoubiquitylated in the absence of damaging agents. Together these data show that Rad7 E3 activity attenuates ubiquitylation of proteins regulating the shuttling of polyubiquitylated proteins to the proteasome (Dsk2 and Rpn10) and removal of ubiquitin chains just prior to degradation (Rpn11). Since Rad7 E3 ligase activity has been shown to increase ubiquitylation of its target proteins, yet our results show increased ubiquitylation in the absence of Rad7 E3, we suggest that Rad7 E3 action regulates ubiquitin ligase and deubiquitylase (DUB) activities that act on Rpn10, Dsk2 and Rpn11.

Impaired DNA Damage Repair Capacity is Associated with an Increased Risk of Esophageal Adenocarcinoma: A Case Control Study

Background: Inherited suboptimal DNA repair capacity in peripheral blood lymphocytes (PBLs) can be unmasked by mutagen challenge and has been associated with susceptibility to cancer. Purpose: To use comet assay to assess the esophageal adenocarcinoma (EAC) risk in relation to mutageninduced DNA damage in PBLs. Materials and methods: In a case-control study, the baseline, benzo[a]pyrene diol epoxide (BPDE)-induced, and γ radiation-induced DNA damage were quantified by the Olive tail moment (TM) in PBLs from 172 Caucasian EAC patients and 154 healthy controls who were frequency matched on age and gender. Logistic regression analysis was used to calculate odds ratios (OR) and 95% confidence intervals (CI) to estimate EAC risk in relation to DNA damage. Results: EAC patients tended to have higher DNA damage than controls, as measured by baseline, net BPDEand net γ radiation-induced TM, but the difference was statistically significant only for net BPDE-induced DNA damage (0.88 ± 0.94 vs. 0.62 ± 0.77, P=0.031). Using the 75th percentile TM in the controls as cutoff point, we found that high levels of net BPDE- and γ radiation-induced DNA damage were associated with significantly increased risks of EAC, with adjusted ORs of 2.15 (95% CI, 1.13-4.10) and 2.27 (95% CI, 1.24–4.16), respectively. EAC risks were further increased among individuals with both net mutagen-induced DNA damages and exposure to gastroesophageal reflux disease or smoking, known risk factors for EAC. Conclusion: Our results suggest impaired repair capacity of mutagen-induced DNA damage in PBLs assessed by comet assay may be a risk factor of EAC.

Effects of Intravenous Benzo[a]Pyrene Dose Administration on Levels of Exposure Biomarkers, DNA Adducts, and Gene Expression in Rats

The effects of benzo[a]pyrene (BaP) administration on biomarkers of exposure and early effects were studied in male Sprague-Dawley rats intravenously injected with doses of 0.4, 4, 10, or 40 μmol BaP/kg . Blood, tissues, and excreta were collected 8 and 24 h posttreatment. BaP and several of its metabolites were simultaneously measured in blood, tissues and excreta by ultra-high-performance liquid chromatography (UHPLC)/fluorescence. DNA adducts of BaP diol epoxide (BaPDE) in lungs were quantified using an ultrasensitive immunoassay with chemiluminescence detection. Expression of selected genes in lungs of treated rats (lung RNA) compared to control rats was also assessed by quantitative real-time polymerase chain reaction. There was a dose-dependent increase in blood, tissue, and excreted levels of BaP metabolites. At 8 and 24 h postinjection, BaP and hydroxyBaP were found in higher concentrations in blood and tissues compared to other analytes. However, diolBaP were excreted in greater amounts in urine and apparently more rapidly than hydroxyBaP. Mean percentages (± SD) of injected dose excreted in urine as 4,5-diolBaP during the 0–8 h and 0–24 h period posttreatment were 0.16 ± 0.027% and 0.14 ± 0.083%, respectively. Corresponding values for 3-OHBaP were 0.0045 ± 0.0009% and 0.026 ± 0.014%. BaP-diones were not detectable in blood, tissues, and excreta; 7,8-diolBaP and BaPtetrol were found to be minor metabolites. There was also a dose-dependent increase in DNA adduct formation in lung. Analysis of gene expression further showed a modulation of Cyp1a1, Cyp1b1, Nqo1, Nrf2, Fos, and Ahr expression at 10- and 40-μmol/kg doses, but not at the lower doses. This study provided a better assessment of the influence of absorbed BaP doses on biological levels of diolBaP and OHBaP exposure biomarkers and association of the latter with early biological alterations, such as DNA adducts and gene expression.

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (PcnaK164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.

Association between individual PM2.5 exposure and DNA damage in traffic policemen.

The aim of this study was to explore the association between individual PM2.5 and DNA damage in traffic policemen. The participants included 110 traffic policemen and 101 common populations. The continuous 24-hour individual PM2.5 measurement was performed in participants. BPDE (benzo[a]pyrene 7,8-diol-9,10-epoxide)-DNA adducts and 1-OHP were detected. The average concentration of 24-hour personal PM2.5 for traffic policemen was significantly higher than that in the control group. PM2.5 exposure is associated with a 1.1% increase in 1-OHP and 0.8% increase in BPDE-DNA adducts after adjusted for body mass index, educational time period, and alcohol intake. Exposure group has 2.04 times higher of 1-OHP and 1.25 times higher of BPDE-DNA adducts when compared to the control group. These results demonstrated that traffic policemen have been a high-risk group suffering DNA damage because of the high PM2.5 exposure.

Association between cigarette smoking and the vaginal microbiota: a pilot study.

BackgroundSmoking has been identified in observational studies as a risk factor for bacterial vaginosis (BV), a condition defined in part by decimation of Lactobacillus spp. The anti-estrogenic effect of smoking and trace amounts of benzo[a]pyrene diol epoxide (BPDE) may predispose women to BV. BPDE increases bacteriophage induction in Lactobacillus spp. and is found in the vaginal secretions of smokers. We compared the vaginal microbiota between smokers and non-smokers and followed microbiota changes in a smoking cessation pilot study.MethodsIn 2010–2011, 20 smokers and 20 non-smokers were recruited to a cross-sectional study (Phase A) and 9 smokers were enrolled and followed for a 12-week smoking cessation program (Phase B). Phase B included weekly behavioral counseling and nicotine patches to encourage smoking cessation. In both phases, participants self-collected mid-vaginal swabs (daily, Phase B) and completed behavioral surveys. Vaginal bacterial composition was characterized by pyrosequencing of barcoded 16S rRNA genes (V1-V3 regions). Vaginal smears were assigned Nugent Gram stain scores. Smoking status was evaluated (weekly, Phase B) using the semi-quantitative NicAlert® saliva cotinine test and carbon monoxide (CO) exhalation.ResultsIn phase A, there was a significant trend for increasing saliva cotinine and CO exhalation with elevated Nugent scores (P value <0.005). Vaginal microbiota clustered into three community state types (CSTs); two dominated by Lactobacillus (L. iners, L. crispatus), and one lacking significant numbers of Lactobacillus spp. and characterized by anaerobes (termed CST-IV). Women who were observed in the low-Lactobacillus CST-IV state were 25-fold more likely to be smokers than those dominated by L. crispatus (aOR: 25.61, 95 % CI: 1.03-636.61). Four women completed Phase B. One of three who entered smoking cessation with high Nugent scores demonstrated a switch from CST-IV to a L.iners-dominated profile with a concomitant drop in Nugent scores which coincided with completion of nicotine patches. The other two women fluctuated between CST-IV and L. iners-dominated CSTs. The fourth woman had low Nugent scores with L. crispatus-dominated CSTs throughout.ConclusionSmokers had a lower proportion of vaginal Lactobacillus spp. compared to non-smokers. Smoking cessation should be investigated as an adjunct to reducing recurrent BV. Larger studies are needed to confirm these findings.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-471) contains supplementary material, which is available to authorized users.

Structural basis for error-free bypass of bulky adduct by DNA polymerase Polκ

Humans are frequently exposed to the environmentally ubiquitous and potentially carcinigenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BP). BP is metabolized to highly reactive benzo[a]pyrene diol epoxides (BPDEs) in the cells. BPDEs react with DNA predominantly at the N2 position of guanine and form bulky adducts. The major BP adduct is (+)-trans-anti-[BP]-N2-dG (BP-N2-dG) that is carcinogenic. The bulky adduct block DNA synthesis by replicative or high-fidelity DNA polymerases. Some of the specialized lesion bypass polymerases (mostly belonging to Y-family) can replicate through this bulky adduct but often in an error prone manner, resulting in mutagenesis. Among the four human Y-family polymerases Polη, Polκ, Polι and Rev1, Polκ is unique in its ability for efficient and error-free replication through BP induced BP-N2-dG adduct. In this study, we determined the crystal structures of human Polκ (hPolκ) in ternary complex with DNA and an incoming nucleotide dCTP analogue. The crystals contain DNA with either G base or (+)-trans-anti-[BP]-N2-dG adduct at a template-primer junction and diffract to 2.5 Å and 2.8 Å, respectively. The structures reveal that hPolκ is able to accommodate the bulky adducted DNA in its minor groove without base flipping and nucleotide looping out. The bulky adduct has the polycyclic BP moiety in the minor groove in the regular helical conformation. Polκ has a unique active site that is more open at the minor groove side than other Y-family polymerases. The damaged guanine is in the anti-conformation, the dCMPNPP incoming nucleotide maintains normal Watson-Crick pairing with the G* base. This is the first structure of eukaryotic Y-family polymerase carrying the minor groove BP adduct. The structure and biochemical analysis provides a basis for understanding how hPolκ can correctly bypass and tolerate BP induced BP-N2-dG adduct in human cells.

Benzo(a)pyrene-7,8-diol-9,10-epoxide induced p53-independent necrosis via the mitochondria-associated pathway involving Bax and Bak activation.

Benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is a highly reactive DNA damage agent and can induce cell death through both p53-independent and -dependent pathways. However, little is known about the molecular mechanisms of p53-independent pathways in BPDE-induced cell death. To understand the p53-independent mechanisms, we have now examined BPDE-induced cytotoxicity in p53-deficient baby mouse kidney (BMK) cells. The results showed that BPDE could induce Bax and Bak activation, cytochrome c release, caspases activation, and necrotic cell death in the BMK cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore, were interdependently activated by BPDE, with Bax and Bak translocation to and Bax/Bak homo-oligomerization in mitochondria, release of cytochrome c was induced. Importantly, cytochrome c release and necrotic cell death were diminished in BMK cells (Bax(-/-)), BMK cells (Bak(-/-)), and BMK cells (Bax(-/-)/Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate BPDE-induced cytochrome c release and necrosis. Together the findings suggested that BPDE-induced necrosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.

Low-Dose Benzo(a)pyrene and Its Epoxide Metabolite Inhibit Myogenic Differentiation in Human Skeletal Muscle-Derived Progenitor Cells

The risk of low birth weights is elevated in prenatal exposure to polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous environmental pollutants generated from combustion of organic compounds, including cigarette smoke. We hypothesized that benzo(a)pyrene (BaP), a member of PAHs existing in cigarette smoke, may affect the myogenesis to cause low birth weights. We investigated the effects of BaP and its main metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), on the myogenic differentiation of human skeletal muscle-derived progenitor cells (HSMPCs). HSMPCs were isolated by a modified preplate technique and cultured in myogenic differentiation media with or without BaP and BPDE (0.25 and 0.5 μM) for 4 days. The multinucleated myotube formation was morphologically analyzed by hematoxylin and eosin staining. The expressions of myogenic differentiation markers and related signaling proteins were determined by Western blotting. Both BaP and BPDE at the submicromolar concentrations (0.25 and 0.5 μM) dose-dependently repressed HSMPCs myogenic differentiation without obvious cell toxicity. Both BaP and BPDE inhibited the muscle-specific protein expressions (myogenin and myosin heavy chain) and phosphorylation of Akt (a known modulator in myogenesis), which could be significantly reversed by the inhibitors for aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and nuclear factor (NF)-κB. BaP- and BPDE-activated NF-κB-p65 protein phosphorylation could also be attenuated by both AhR and ER inhibitors. The inhibitory effects of BaP and BPDE on myogenesis were reversed after withdrawing BaP exposure, but not after BPDE withdrawal. These results suggest that both BaP and BPDE are capable of inhibiting myogenesis via an AhR- or/and ER-regulated NF-κB/Akt signaling pathway.

Benzo[a]pyrene increases DNA double strand break repair in vitro and in vivo: A possible mechanism for benzo[a]pyrene-induced toxicity

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon and carcinogen that is released into the environment through natural and anthropogenic sources. BaP toxicity is dependent on its metabolism by cytochrome P450s to the reactive metabolite benzo[a]pyrene diol epoxide (BPDE), which is strongly associated with increased mutation frequency. BaP can also be metabolized to benzo[a]pyrene quinones that can undergo redox cycling and induce oxidative stress. The purpose of this study was to examine if BaP exposure induces DNA double strand breaks (DSBs) and subsequently activate DNA DSB repair pathways in the CHO 3–6 cell line and pKZ1 mouse model. In vitro assessment of homologous recombination (HR) showed significantly increased HR frequency following exposure to 10μM of BaP. In vivo evaluations of BaP-induced DNA DSB repair demonstrated positive staining for intrachromosomal recombination events, which are associated with non-homologous end joining (NHEJ), in the lung and thymus of exposed animals that were statistically significant in the thymus when quantified by Western blotting. Gene expression analyses from mouse tissues showed significantly decreased expression of ATM and Xrcc6 in BaP-treated liver and lung. In addition, BaP exposure significantly reduced the expression of Xrcc5, p53, and DNA-PKcs in lung. Taken together, our results demonstrate that BaP increases DNA DSB repair in vitro and in vivo, and induces expression changes in DNA repair pathway genes. As repair of DNA DSBs is not error-free, aberrant DNA repair may be contributing to the mechanism of BaP-induced toxicity.

Oxidative damage to nucleic acids and benzo(a)pyrene-7,8-diol-9,10-epoxide-DNA adducts and chromosomal aberration in children with psoriasis repeatedly exposed to crude coal tar ointment and UV radiation.

The paper presents a prospective cohort study. Observed group was formed of children with plaque psoriasis (n=19) treated by Goeckerman therapy (GT). The study describes adverse (side) effects associated with application of GT (combined exposure of 3% crude coal tar ointment and UV radiation). After GT we found significantly increased markers of oxidative stress (8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, and 8-hydroxyguanine), significantly increased levels of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) DNA adducts (BPDE-DNA), and significantly increased levels of total number of chromosomal aberrations in peripheral lymphocytes. We found significant relationship between (1) time of UV exposure and total number of aberrated cells and (2) daily topical application of 3% crude coal tar ointment (% of body surface) and level of BPDE-DNA adducts. The findings indicated increased hazard of oxidative stress and genotoxic effects related to the treatment. However, it must be noted that the oxidized guanine species and BPDE-DNA adducts also reflect individual variations in metabolic enzyme activity (different extent of bioactivation of benzo[a]pyrene to BPDE) and overall efficiency of DNA/RNA repair system. The study confirmed good effectiveness of the GT (significantly decreased PASI score).

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.

Abstract CN02-01: Factors that impinge on the p53 pathway modify cancer risk

Abstract Mdm2 and Mdm4 are critical inhibitors of the tumor suppressor p53 and keep p53 activity at bay under normal physiological conditions. Many tumors thus inactivate the p53 pathway by over producing Mdm2 and/or Mdm4. A single nucleotide polymorphism (SNP) in Mdm2 was recently identified that associates with increased levels of Mdm2 and increased risk of breast cancer in Li-Fraumeni syndrome patients. To directly test the role of SNP309 in cancer risk, we generated SNP mice and show that mice inheriting two copies of SNP309G are at increased risk of developing tumors in normal mice compared to SNP309T homozygous mice. To examine the effects of environmental factors and carcinogens, we treated these mice with a low dose of ionizing radiation or with Benzo[a]pyrene-7,8-diol-9,10-epoxide (BaP) a carcinogen found in tobacco smoke. Radiation increased the risk of cancers in SNP309G mice. However, in the skin model of BaP, the SNP309T allele conferred increased risk of papillomas. The data suggest that factors that regulate Mdm2 levels vary between different tissues and ultimately impinge on the risk of tumor development. Citation Format: Guillermina Lozano. Factors that impinge on the p53 pathway modify cancer risk. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr CN02-01.

Inhibition of HIV-1 Reverse Transcriptase-Catalyzed Synthesis by Intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide Adducts

To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N 6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

Inter-individual variation in nucleotide excision repair pathway is modulated by non-synonymous polymorphisms in ERCC4 and MBD4 genes

Inter-individual differences in DNA repair capacity (DRC) may lead to genome instability and, consequently, modulate individual cancer risk. Among the different DNA repair pathways, nucleotide excision repair (NER) is one of the most versatile, as it can eliminate a wide range of helix-distorting DNA lesions caused by ultraviolet light irradiation and chemical mutagens. We performed a genotype–phenotype correlation study in 122 healthy subjects in order to assess if any associations exist between phenotypic profiles of NER and DNA repair gene single nucleotide polymorphisms (SNPs). Individuals were genotyped for 768 SNPs with a custom Illumina Golden Gate Assay, and peripheral blood mononuclear cells (PBMCs) of the same subjects were tested for a NER comet assay to measure DRC after challenging cells by benzo(a)pyrene diolepoxide (BPDE).We observed a large inter-individual variability of NER capacity, with women showing a statistically significant lower DRC (mean±SD: 6.68±4.76; p=0.004) than men (mean±SD: 8.89±5.20). Moreover, DRC was significantly lower in individuals carrying a variant allele for the ERCC4 rs1800124 non-synonymous SNP (nsSNP) (p=0.006) and significantly higher in subjects with the variant allele of MBD4 rs2005618 SNP (p=0.008), in linkage disequilibrium (r2=0.908) with rs10342 nsSNP.Traditional in silico docking approaches on protein–DNA and protein–protein interaction showed that Gly875 variant in ERCC4 (rs1800124) decreases the DNA–protein interaction and that Ser273 and Thr273 variants in MBD4 (rs10342) indicate complete loss of protein–DNA interactions. Our results showed that NER inter-individual capacity can be modulated by cross-talk activity involving nsSNPs in ERCC4 and MBD4 genes, and they suggested to better investigate SNP effect on cancer risk and response to chemo- and radiotherapies.

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.

Sequence context modulation of polycyclic aromatic hydrocarbon‐induced mutagenesis

DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N(6) -BPDE dA adducts at the second position in the N-ras codon 61(CAA) (61(2) ) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10 S-BPDE and C10 R-BPDE lesions linked through the N(6) position of adenine in the sequence context N-ras codon 61, position 3 (C10 S-BPDE and C10 R-BPDE at 61(3) ) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10 S-BPDE and C10 R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 61(3) resulted in truncated products, with the C10 S-BPDE adducts being more blocking than C10 R-BPDE lesions, and termination of synthesis was more pronounced at position 61(3) than at 61(2) for each of the lesions.

Association between genetic polymorphisms in pre-miR-146a and pre-miR-196a2 and genetic damage levels among coke oven workers

To investigate the association of rs2910164 G > C polymorphism and rs11614913 T > C polymorphism in pre-miR-146a and pre-miR-196a2 with genetic damage levels in coke oven workers. A total of 575 nonsmoking workers who have worked for more than one year in a coke-oven plant at Wuhan, Hubei Province were enrolled in this study in September to October, 2010. The general characteristics as well as blood and urine samples were collected. The genetic damage levels were detected by cytokinesis-block micronucleus cytom assay and represented as micronucleus (MN) frequencies of binucleate cells in peripheral blood lymphocytes. The rs2910164 G > C polymorphisms in pre-miR-146a and rs11614913 T > C polymorphisms in pre-miR-196a2 were genotyped by using TaqMan assay. The plasma concentrations of benzo[a]pyrene-diolepoxide (BPDE)-albumin adducts were determined by using ELISA. All data were analyzed, the frequency ratio (FR) and 95%CI were calculated. Totally, 575 workers were taken into consideration. The rs2910164 C allele was associated with increased MN frequencies in the coke oven workers (P trend = 0.025), and the MN frequencies were higher in rs2910164 CC genotype carriers (4.38 ± 3.46) than in wild-type rs2910164 GG genotype carriers (4.02 ± 3.09) (FR = 1.18, 95%CI:1.04-1.34). The further stratified analyses by working years, gender, alcohol consumption, and the levels of BPDE-albumin adducts showed that the effects of rs2910164 C allele in increasing MN frequencies were robust in subjects who were males (FR = 1.11, 95%CI:1.02-1.20), nondrinkers (FR = 1.07, 95%CI:1.00-1.14), working years less than 20 (FR = 1.12, 95%CI:1.03-1.22), and workers with lower BPDE-albumin adducts levels (FR = 1.11, 95%CI:1.02-1.21) (P trend = 0.011, 0.044, 0.006 and 0.020, respectively). In addition, the MN frequencies were higher in workers with rs11614913 TC genotype (4.27 ± 2.91) than workers with rs11614913 TT genotype (3.90 ± 3.32) (FR = 1.12, 95%CI:1.02-1.23).Workers carried both rs2910164 GG and rs11614913 TT genotypes were set as a control, and the MN frequencies of workers with both rs2910164 CC and rs11614913 CC genotypes (5.32 ± 4.94) were 1.51 (1.21-1.89) times higher than the control (3.75 ± 3.01). The rs2910164 C allele in pre-miR-146a and rs11614913 C allele in pre-miR-196a2 were associated with increased genetic damage levels in coke oven workers.