Dataset Information

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene.

ABSTRACT: Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is the major cause of lung cancer. BaP forms covalent DNA adducts after metabolic activation and induces mutations. We have developed a method for capturing oligonucleotides carrying bulky base adducts, including UV-induced cyclobutane pyrimidine dimers (CPDs) and BaP diol epoxide-deoxyguanosine (BPDE-dG), which are removed from the genome by nucleotide excision repair. The isolated oligonucleotides are ligated to adaptors, and after damage-specific immunoprecipitation, the adaptor-ligated oligonucleotides are converted to dsDNA with an appropriate translesion DNA synthesis (TLS) polymerase, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method translesion excision repair-sequencing (tXR-seq). In contrast to our previously described XR-seq method, tXR-seq does not depend on repair/removal of the damage in the excised oligonucleotides, and thus it is applicable to essentially all DNA damages processed by nucleotide excision repair. Here we present the excision repair maps for CPDs and BPDE-dG adducts generated by tXR-Seq for the human genome. In addition, we report the sequence specificity of BPDE-dG excision repair using tXR-seq.

SUBMITTER: Li W

PROVIDER: S-EPMC5495276 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene.

Li Wentao W Hu Jinchuan J Adebali Ogun O Adar Sheera S Yang Yanyan Y Chiou Yi-Ying YY Sancar Aziz A

Proceedings of the National Academy of Sciences of the United States of America 20170612 26

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is the major cause of lung cancer. BaP forms covalent DNA adducts after metabolic activation and induces mutations. We have developed a method for capturing oligonucleotides carrying bulky base adducts, including UV-induced cyclobutane pyrimidine dimers (CPDs) and BaP diol epoxide-deoxyguanosine (BPDE-dG), which are removed from the genome by nucleotide excision repair. The isolated oligonucleotides are ligated to adaptors, and after damag ...[more]

PMID: 28607059

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Project description:BackgroundBenzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown.Methodology/principal findingsUsing neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels.Conclusions/significanceContrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today's perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on other health hazards that such pollutants may exert.

Project description:In the development of new chemical substances, genetic toxicity evaluations are a high priority for safety risk management. Evaluation of the possibility of compound carcinogenicity with accuracy and at reasonable cost in the early stages of development by in vitro techniques is preferred. Currently, DNA damage-related in vitro genotoxicity tests are widely-used screening tools after which next generation toxicity testing may be applied to confirm DNA damage. DNA adductomics may be used to evaluate DNA damage in vitro, however confirmation of DNA adduct identities through comparison to authentic standards may be time-consuming and expensive processes. Considering this, a streamlined method for confirming putative DNA adducts that are detected by DNA adductomics may be useful. With this aim, in vitro DNA adductome methods in conjunction with in vitro RNA adductome methods may be proposed as a DNA adductome verification approach by which to eliminate false positive annotations. Such an approach was evaluated by conducting in vitro assays whereby Hep G2 cell lines that were exposed to or not exposed to benzo[a]pyrene were digested to their respective 2'-deoxynucleosides or ribonucleosides and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) by comparative DNA and RNA adductomics through neutral loss targeting of the [M + H]+ > [M + H - 116]+ or [M + H]+ > [M + H -132]+ transitions over predetermined ranges. Comparisons of DNA adductome maps revealed putative DNA adducts that were detected in exposed cells but not in unexposed cells. Similarly, comparisons of RNA adductome maps revealed putative RNA adducts in exposed cells but not in unexposed cells. Taken together these experiments revealed that analogous forms of putative damage had occurred in both DNA and RNA which supported that putative DNA adducts detected by DNA adductomics were DNA adducts. High resolution mass spectrometry (HRMS) was utilized to confirm that putative nucleic acid adducts detected in both DNA and RNA were derived from benzo[a]pyrene exposure and these putative adducts were identified as 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene- (B[a]PDE)-type adducts. Overall, this study demonstrates the usefulness of utilizing DNA/RNA adductomics to screen for nucleic acid damage.

Dataset Information

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene.

Publications

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene.

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