RESUMO
Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.
Assuntos
Membrana Celular/metabolismo , Polissacarídeos/metabolismo , RNA/metabolismo , Animais , Anticorpos/metabolismo , Sequência de Bases , Vias Biossintéticas , Linhagem Celular , Sobrevivência Celular , Humanos , Espectrometria de Massas , Ácido N-Acetilneuramínico/metabolismo , Poliadenilação , Polissacarídeos/química , RNA/química , RNA/genética , RNA não Traduzido/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Coloração e RotulagemRESUMO
The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin's biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis.
Assuntos
Microbioma Gastrointestinal , Policetídeos , Humanos , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Escherichia coli/metabolismo , Policetídeos/química , Peptídeo Hidrolases/químicaRESUMO
Endogenous electrophiles, ionizing and non-ionizing radiation, and hazardous chemicals present in the environment and diet can damage DNA by forming covalent adducts. DNA adducts can form in critical cancer driver genes and, if not repaired, may induce mutations during cell division, potentially leading to the onset of cancer. The detection and quantification of specific DNA adducts are some of the first steps in studying their role in carcinogenesis, the physiological conditions that lead to their production, and the risk assessment of exposure to specific genotoxic chemicals. Hundreds of different DNA adducts have been reported in the literature, and there is a critical need to establish a DNA adduct mass spectral database to facilitate the detection of previously observed DNA adducts and characterize newly discovered DNA adducts. We have collected synthetic DNA adduct standards from the research community, acquired MSn (n = 2, 3) fragmentation spectra using Orbitrap and Quadrupole-Time-of-Flight (Q-TOF) MS instrumentation, processed the spectral data and incorporated it into the MassBank of North America (MoNA) database, and created a DNA adduct portal Web site (https://sites.google.com/umn.edu/dnaadductportal) to serve as a central location for the DNA adduct mass spectra and metadata, including the spectral database downloadable in different formats. This spectral library should prove to be a valuable resource for the DNA adductomics community, accelerating research and improving our understanding of the role of DNA adducts in disease.
Assuntos
Adutos de DNA , DNA , Humanos , DNA/química , Espectrometria de Massas , Dano ao DNA , CarcinogêneseRESUMO
Air pollution, tobacco smoke, and red meat are associated with renal cell cancer (RCC) risk in the United States and Western Europe; however, the chemicals that form DNA adducts and initiate RCC are mainly unknown. Aristolochia herbaceous plants are used for medicinal purposes in Asia and worldwide. They are a significant risk factor for upper tract urothelial carcinoma (UTUC) and RCC to a lesser extent. The aristolochic acid (AA) 8-methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I), a component of Aristolochia herbs, contributes to UTUC in Asian cohorts and in Croatia, where AA-I exposure occurs from ingesting contaminated wheat flour. The DNA adduct of AA-I, 7-(2'-deoxyadenosin-N6-yl)-aristolactam I, is often detected in patients with UTUC, and its characteristic A:T-to-T:A mutational signature occurs in oncogenes and tumor suppressor genes in AA-associated UTUC. Identifying DNA adducts in the renal parenchyma and pelvis caused by other chemicals is crucial to gaining insights into unknown RCC and UTUC etiologies. We employed untargeted screening with wide-selected ion monitoring tandem mass spectrometry (wide-SIM/MS2) with nanoflow liquid chromatography/Orbitrap mass spectrometry to detect DNA adducts formed in rat kidneys and liver from a mixture of 13 environmental, tobacco, and dietary carcinogens that may contribute to RCC. Twenty DNA adducts were detected. DNA adducts of 3-nitrobenzanthrone (3-NBA), an atmospheric pollutant, and AA-I were the most abundant. The nitrophenanthrene moieties of 3-NBA and AA-I undergo reduction to their N-hydroxy intermediates to form 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts. We also discovered a 2'-deoxycytidine AA-I adduct and dA and dG adducts of 10-methoxy-6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-III), an AA-I isomer and minor component of the herbal extract assayed, signifying AA-III is a potent kidney DNA-damaging agent. The roles of AA-III, other nitrophenanthrenes, and nitroarenes in renal DNA damage and human RCC warrant further study. Wide-SIM/MS2 is a powerful scanning technology in DNA adduct discovery and cancer etiology characterization.
Assuntos
Ácidos Aristolóquicos , Carcinoma de Células Renais , Carcinoma de Células de Transição , Neoplasias Renais , Neoplasias da Bexiga Urinária , Ratos , Animais , Humanos , Adutos de DNA , Carcinoma de Células Renais/patologia , Carcinoma de Células de Transição/patologia , Farinha/análise , Neoplasias da Bexiga Urinária/patologia , Triticum , Ácidos Aristolóquicos/química , DNA , Rim/patologia , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/patologia , Fígado/química , Ácidos Carboxílicos , Carcinógenos/químicaRESUMO
Exogenous compounds and metabolites derived from therapeutics, microbiota, or environmental exposures directly interact with endogenous metabolic pathways, influencing disease pathogenesis and modulating outcomes of clinical interventions. With few spectral library references, the identification of covalently modified biomolecules, secondary metabolites, and xenobiotics is a challenging task using global metabolomics profiling approaches. Numerous liquid chromatography-coupled mass spectrometry (LC-MS) small molecule analytical workflows have been developed to curate global profiling experiments for specific compound groups of interest. These workflows exploit shared structural moiety, functional groups, or elemental composition to discover novel and undescribed compounds through nontargeted small molecule discovery pipelines. This Review introduces the concept of structure-oriented LC-MS discovery methodology and aims to highlight common approaches employed for the detection and characterization of covalently modified biomolecules, secondary metabolites, and xenobiotics. These approaches represent a combination of instrument-dependent and computational techniques to rapidly curate global profiling experiments to detect putative ions of interest based on fragmentation patterns, predictable phase I or phase II metabolic transformations, or rare elemental composition. Application of these methods is explored for the detection and identification of novel and undescribed biomolecules relevant to the fields of toxicology, pharmacology, and drug discovery. Continued advances in these methods expand the capacity for selective compound discovery and characterization that promise remarkable insights into the molecular interactions of exogenous chemicals with host biochemical pathways.
Assuntos
Espectrometria de Massas em Tandem , Xenobióticos , Cromatografia Líquida , Descoberta de Drogas , Exposição AmbientalRESUMO
DNA alkylating drugs have been used as frontline medications to treat cancer for decades. Their chemical reaction with DNA leads to the blockage of DNA replication, which impacts cell replication. While this impacts rapidly dividing cancerous cells, this process is not selective and results in highly variable and often severe side effects in patients undergoing alkylating-drug based therapies. The development of biomarkers to identify patients who effectively respond with tolerable toxicities vs patients who develop serious side effects is needed. Cyclophosphamide (CPA) is a commonly used chemotherapeutic drug and lacks biomarkers to evaluate its therapeutic effect and toxicity. Upon administration, CPA is metabolically activated and converted to phosphoramide mustard and acrolein, which are responsible for its efficacy and toxicity, respectively. Previous studies have explored the detection of the major DNA adduct of CPA, the interstrand DNA-DNA cross-link G-NOR-G, finding differences in the cross-link amount between Fanconi Anemia and non-Fanconi Anemia patients undergoing chemotherapy treatment. In this study, we take advantage of our DNA adductomic approach to comprehensively profile CPA's and its metabolites' reactions with DNA in vitro and in patients undergoing CPA-based chemotherapy. This investigation led to the detection of 40 DNA adducts in vitro and 20 DNA adducts in patients treated with CPA. Moreover, acrolein-derived DNA adducts were quantified in patient samples. The results suggest that CPA-DNA damage is very complex, and an evaluation of DNA adduct profiles is necessary when evaluating the relationship between CPA-DNA damage and patient outcome.
Assuntos
Anemia , Adutos de DNA , Humanos , Acroleína/farmacologia , Ciclofosfamida/efeitos adversos , Alquilantes , Dano ao DNA , DNA/química , Espectrometria de Massas , Cromatografia LíquidaRESUMO
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco products, and exposure to it is likely one of the factors contributing to the development of lung cancer in cigarette smokers. To exert its carcinogenic effects, NNK must be metabolically activated into highly reactive species generating a wide spectrum of DNA damage. We have identified a new class of DNA adducts, DNA-RNA cross-links found for the first time in NNK-treated mice lung DNA using our improved high-resolution accurate mass segmented full scan data-dependent neutral loss MS3 screening strategy. The levels of these DNA-RNA cross-links were found to be significantly higher in NNK-treated mice compared to the corresponding controls, which is consistent with higher levels of formaldehyde due to NNK metabolism as compared to endogenous levels. We hypothesize that this DNA-RNA cross-linking occurs through reaction with NNK-generated formaldehyde and speculate that this phenomenon has broad implications for NNK-induced carcinogenesis. The structures of these cross-links were characterized using high-resolution LC-MS2 and LC-MS3 accurate mass spectral analysis and comparison to a newly synthesized standard. Taken together, our data demonstrate a previously unknown link between DNA-RNA cross-link adducts and NNK and provide a unique opportunity to further investigate how these novel NNK-derived DNA-RNA cross-links contribute to carcinogenesis in the future.
Assuntos
Carcinogênese , RNA , Camundongos , Animais , Carcinogênese/induzido quimicamente , Transformação Celular Neoplásica , DNA , Formaldeído/toxicidade , Camundongos Endogâmicos , PulmãoRESUMO
Many chemotherapeutic drugs exert their cytotoxicity through the formation of DNA modifications (adducts), which interfere with DNA replication, an overactive process in rapidly dividing cancer cells. Side effects from the therapy are common, however, because these drugs also affect rapidly dividing noncancerous cells. Hypoxia-activated prodrugs (HAPs) have been developed to reduce these side effects as they preferentially activate in hypoxic environments, a hallmark of solid tumors. CP-506 is a newly developed DNA-alkylating HAP designed to exert strong activity under hypoxia. The resulting CP-506-DNA adducts can be used to elucidate the cellular and molecular effects of CP-506 and its selectivity toward hypoxic conditions. In this study, we characterize the profile of adducts resulting from the reaction of CP-506 and its metabolites CP-506H and CP-506M with DNA. A total of 39 putative DNA adducts were detected in vitro using our high-resolution/accurate-mass (HRAM) liquid chromatography tandem mass spectrometry (LC-MS3) adductomics approach. Validation of these results was achieved using a novel strategy involving 15N-labeled DNA. A targeted MS/MS approach was then developed for the detection of the 39 DNA adducts in five cancer cell lines treated with CP-506 under normoxic and hypoxic conditions to evaluate the selectivity toward hypoxia. Out of the 39 DNA adducts initially identified, 15 were detected, with more adducts observed from the two reactive metabolites and in cancer cells treated under hypoxia. The presence of these adducts was then monitored in xenograft mouse models bearing MDA-MB-231, BT-474, or DMS114 tumors treated with CP-506, and a relative quantitation strategy was used to compare the adduct levels across samples. Eight adducts were detected in all xenograft models, and MDA-MB-231 showed the highest adduct levels. These results suggest that CP-506-DNA adducts can be used to better understand the mechanism of action and monitor the efficacy of CP-506 in vivo, as well as highlight a new role of DNA adductomics in supporting the clinical development of DNA-alkylating drugs.
Assuntos
Adutos de DNA/análise , DNA Bacteriano/análise , DNA/análise , Hipóxia/tratamento farmacológico , Pró-Fármacos/química , Animais , Bovinos , Feminino , Humanos , Hipóxia/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Células Tumorais CultivadasRESUMO
Well-done cooked red meat consumption is linked to aggressive prostate cancer (PC) risk. Identifying mutation-inducing DNA adducts in the prostate genome can advance our understanding of chemicals in meat that may contribute to PC. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic aromatic amine (HAA) formed in cooked meat, is a potential human prostate carcinogen. PhIP was measured in the hair of PC patients undergoing prostatectomy, bladder cancer patients under treatment for cystoprostatectomy, and patients treated for benign prostatic hyperplasia (BPH). PhIP hair levels were above the quantification limit in 123 of 205 subjects. When dichotomizing prostate pathology biomarkers, the geometric mean PhIP hair levels were higher in patients with intermediate and elevated-risk prostate-specific antigen values than lower-risk values <4 ng/mL (p = 0.03). PhIP hair levels were also higher in patients with intermediate and high-risk Gleason scores ≥7 compared to lower-risk Gleason score 6 and BPH patients (p = 0.02). PC patients undergoing prostatectomy had higher PhIP hair levels than cystoprostatectomy or BPH patients (p = 0.02). PhIP-DNA adducts were detected in 9.4% of the patients assayed; however, DNA adducts of other carcinogenic HAAs, and benzo[a]pyrene formed in cooked meat, were not detected. Prostate specimens were also screened for 10 oxidative stress-associated lipid peroxidation (LPO) DNA adducts. Acrolein 1,N2-propano-2'-deoxyguanosine adducts were detected in 54.5% of the patients; other LPO adducts were infrequently detected. Acrolein adducts were not associated with prostate pathology biomarkers, although DNA adductomic profiles differed between PC patients with low and high-grade Gleason scores. Many DNA adducts are of unknown origin; however, dG adducts of formaldehyde and a series of purported 4-hydroxy-2-alkenals were detected at higher abundance in a subset of patients with elevated Gleason scores. The PhIP hair biomarker and DNA adductomics data support the paradigm of well-done cooked meat and oxidative stress in aggressive PC risk.
Assuntos
Hiperplasia Prostática , Neoplasias da Próstata , Acroleína , Biomarcadores , Carcinógenos/análise , DNA , Adutos de DNA , Cabelo/química , Humanos , Masculino , Carne/efeitos adversos , Carne/análise , Piridinas , Dosímetros de RadiaçãoRESUMO
Development of high-resolution/accurate mass liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) methodology enables the characterization of covalently modified DNA induced by interaction with genotoxic agents in complex biological samples. Constant neutral loss monitoring of 2'-deoxyribose or the nucleobases using data-dependent acquisition represents a powerful approach for the unbiased detection of DNA modifications (adducts). The lack of available bioinformatics tools necessitates manual processing of acquired spectral data and hampers high throughput application of these techniques. To address this limitation, we present an automated workflow for the detection and curation of putative DNA adducts by using diagnostic fragmentation filtering of LC-MS/MS experiments within the open-source software MZmine. The workflow utilizes a new feature detection algorithm, DFBuilder, which employs diagnostic fragmentation filtering using a user-defined list of fragmentation patterns to reproducibly generate feature lists for precursor ions of interest. The DFBuilder feature detection approach readily fits into a complete small-molecule discovery workflow and drastically reduces the processing time associated with analyzing DNA adductomics results. We validate our workflow using a mixture of authentic DNA adduct standards and demonstrate the effectiveness of our approach by reproducing and expanding the results of a previously published study of colibactin-induced DNA adducts. The reported workflow serves as a technique to assess the diagnostic potential of novel fragmentation pattern combinations for the unbiased detection of chemical classes of interest.
Assuntos
Adutos de DNA , Espectrometria de Massas em Tandem , Cromatografia Líquida , DNA , SoftwareRESUMO
A novel software has been created to comprehensively characterize covalent modifications of DNA through mass spectral analysis of enzymatically hydrolyzed DNA using the neutral loss of 2'-deoxyribose, a nearly universal MS2 fragmentation process of protonated 2'-deoxyribonucleosides. These covalent modifications termed DNA adducts form through xenobiotic exposures or by reaction with endogenous electrophiles and can induce mutations during cell division and initiate carcinogenesis. DNA adducts are typically present at trace levels in the human genome, requiring a very sensitive and comprehensive data acquisition and analysis method. Our software, wSIM-City, was created to process mass spectral data acquired by a wide selected ion monitoring (wSIM) with gas-phase fractionation and coupled to wide MS2 fragmentation. This untargeted approach can detect DNA adducts at trace levels as low as 1.5 adducts per 109 nucleotides. This level of sensitivity is sufficient for comprehensive analysis and characterization of DNA modifications in human specimens.
Assuntos
Adutos de DNA , DNA , Espectrometria de Massas por Ionização por Electrospray , Humanos , Espectrometria de Massas , Nucleotídeos , XenobióticosRESUMO
BACKGROUND: Both human and veterinary cancer chemotherapy are undergoing a paradigm shift from a "one size fits all" approach to more personalized, patient-oriented treatment strategies. Personalized chemotherapy is dependent on the identification and validation of biomarkers that can predict treatment outcome and/or risk of toxicity. Many cytotoxic chemotherapy agents, including doxorubicin, base their mechanism of action by interaction with DNA and disruption of normal cellular processes. We developed a high-resolution/accurate-mass liquid chromatography-mass spectrometry DNA screening approach for monitoring doxorubicin-induced DNA modifications (adducts) in vitro and in vivo. We used, for the first time, a new strategy involving the use of isotope-labeled DNA, which greatly facilitates adduct discovery. The overall goal of this work was to identify doxorubicin-DNA adducts to be used as biomarkers to predict drug efficacy for use in veterinary oncology. RESULTS: We used our novel mass spectrometry approach to screen for adducts in purified DNA exposed to doxorubicin. This initial in vitro screening identified nine potential doxorubicin-DNA adduct masses, as well as an intense signal corresponding to DNA-intercalated doxorubicin. Two of the adduct masses, together with doxorubicin and its metabolite doxorubicinol, were subsequently detected in vivo in liver DNA extracted from mice exposed to doxorubicin. Finally, the presence of these adducts and analytes was explored in the DNA isolated from dogs undergoing treatment with doxorubicin. The previously identified nine DOX-DNA adducts were not detected in these preliminary three samples collected seven days post-treatment, however intercalated doxorubicin and doxorubicinol were detected. CONCLUSIONS: This work sets the stage for future evaluation of doxorubicin-DNA adducts and doxorubicin-related molecules as candidate biomarkers to personalize chemotherapy protocols for canine cancer patients. It demonstrates our ability to combine in one method the analysis of DNA adducts and DNA-intercalated doxorubicin and doxorubicinol. The last two analytes interestingly, were persistent in samples from canine patients undergoing doxorubicin chemotherapy seven days after treatment. The presence of doxorubicin in all samples suggests a role for it as a promising biomarker for use in veterinary chemotherapy. Future studies will involve the analysis of more samples from canine cancer patients to elucidate optimal timepoints for monitoring intercalated doxorubicin and doxorubicin-DNA adducts and the correlation of these markers with therapy outcome.
Assuntos
Doenças do Cão , Doxorrubicina , Neoplasias , Animais , Biomarcadores , DNA , Adutos de DNA , Doenças do Cão/tratamento farmacológico , Cães , Doxorrubicina/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/veterináriaRESUMO
Inhibiting the disease progression in KRAS-driven cancers after diagnosis has been a difficult task for clinicians to manage due to the lack of effective intervention/preventive therapies. KRAS-driven cancers depend on sustained KRAS signaling. Although developing inhibitors of KRAS signaling has proven difficult in the past, the quest for identifying newer agents has not stopped. Based on studies showing terpenoids as modulators of KRAS-regulated downstream molecular pathways, we asked if this chemical family has an affinity of inhibiting KRAS protein activity. Using crystal structure as a bait in silico, we identified 20 terpenoids for their KRAS protein-binding affinity. We next carried out biological validation of in silico data by employing in situ, in vitro, patient-derived explant ex vivo, and KPC transgenic mouse models. In this report, we provide a comprehensive analysis of a lup-20(29)-en-3b-ol (lupeol) as a KRAS inhibitor. Using nucleotide exchange, isothermal titration calorimetry, differential scanning fluorimetry, and immunoprecipitation assays, we show that lupeol has the potential to reduce the guanosine diphosphate/guanosine triphosphate exchange of KRAS protein including mutant KRASG12V . Lupeol treatment inhibited the KRAS activation in KRAS-activated cell models (NIH-panel, colorectal, lung, and pancreatic intraepithelial neoplasia) and patient tumor explants ex vivo. Lupeol reduced the three-dimensional growth of KRAS-activated cells. The pharmacokinetic analysis showed the bioavailability of lupeol after consumption via oral and intraperitoneal routes in animals. Tested under prevention settings, the lupeol consumption inhibited the development of pancreatic intraepithelial neoplasia in LSL-KRASG12D/Pdx-cre mice (pancreatic ductal adenocarcinoma progression model). These data suggest that the selected members of the triterpene family (such as lupeol) could be exploited as clinical agents for preventing the disease progression in KRAS-driven cancers which however warrants further investigation.
Assuntos
Anti-Inflamatórios/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Modelos Animais de Doenças , Neoplasias Pancreáticas/tratamento farmacológico , Triterpenos Pentacíclicos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Apoptose , Proliferação de Células , Transformação Celular Neoplásica/patologia , Progressão da Doença , Feminino , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Mass spectrometry-based DNA adductomics is an emerging approach for the human biomonitoring of hazardous chemicals. A mass spectral database of DNA adducts will be created for the scientific community to investigate the associations between chemical exposures, DNA damage, and disease risk.
Assuntos
Adutos de DNA/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Poluentes Ambientais/farmacologia , Compostos Orgânicos/farmacologia , Dano ao DNA , Poluentes Ambientais/química , Humanos , Espectrometria de Massas , Compostos Orgânicos/químicaRESUMO
African American (AA) smokers are at a higher risk of developing lung cancer compared to whites. The variations in the metabolism of nicotine and tobacco-derived carcinogens in these groups were reported previously with the levels of nicotine metabolites and carcinogen-derived metabolites measured using targeted approaches. While useful, these targeted strategies are not able to detect global metabolic changes for use in predicting the detrimental effects of tobacco use and ultimately lung cancer susceptibility among smokers. To address this limitation, we have performed global untargeted metabolomics profiling in urine of AA and white smokers to characterize the pattern of metabolites, identify differentially regulated pathways, and correlate these profiles with the observed variations in lung cancer risk between these two populations. Urine samples from AA (n = 30) and white (n = 30) smokers were used for metabolomics analysis acquired in both positive and negative electrospray ionization modes. LC-MS data were uploaded onto the cloud-based XCMS online (http://xcmsonline.scripps.edu) platform for retention time correction, alignment, feature detection, annotation, statistical analysis, data visualization, and automated systems biology pathway analysis. The latter identified global differences in the metabolic pathways in the two groups including the metabolism of carbohydrates, amino acids, nucleotides, fatty acids, and nicotine. Significant differences in the nicotine degradation pathway (cotinine glucuronidation) in the two groups were observed and confirmed using a targeted LC-MS/MS approach. These results are consistent with previous studies demonstrating AA smokers with lower glucuronidation capacity compared to whites. Furthermore, the d-glucuronate degradation pathway was found to be significantly different between the two populations, with lower amounts of the putative metabolites detected in AA compared to whites. We hypothesize that the differential regulation of the d-glucuronate degradation pathway is a consequence of the variations in the glucuronidation capacity observed in the two groups. Other pathways including the metabolism of amino acids, nucleic acids, and fatty acids were also identified, however, the biological relevance and implications of these differences across ethnic groups need further investigation. Overall, the applied metabolomics approach revealed global differences in the metabolic networks and endogenous metabolites in AA and whites, which could be used and validated as a new potential panel of biomarkers that could be used to predict lung cancer susceptibility among smokers in population-based studies.
Assuntos
Neoplasias Pulmonares/metabolismo , Metabolômica , Nicotina/metabolismo , Adulto , Cromatografia Líquida , Etnicidade , Humanos , Neoplasias Pulmonares/urina , Pessoa de Meia-Idade , Estrutura Molecular , Nicotina/análise , Fatores de Risco , Fumantes , Espectrometria de Massas em TandemRESUMO
The formation of methyl DNA adducts is a critical step in carcinogenesis initiated by the exposure to methylating carcinogens. Methyl DNA phosphate adducts, formed by methylation of the oxygen atoms of the DNA phosphate backbone, have been detected in animals treated with methylating carcinogens. However, detection of these adducts in human tissues has not been reported. We developed an ultrasensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for detecting methyl DNA phosphate adducts. Using 50 µg of human lung DNA, a limit of quantitation of two adducts/1010 nucleobases was achieved. Twenty-two structurally unique methyl DNA phosphate adducts were detected in human lung DNA. The adduct levels were measured in both tumor and adjacent normal tissues from 30 patients with lung cancer, including 13 current smokers and 17 current non-smokers, as confirmed by measurements of urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Levels of total methyl DNA phosphate adducts in normal lung tissues were higher in smokers than non-smokers, with an average of 13 and 8 adducts/109 nucleobases, respectively. Methyl DNA phosphate adducts were also detected in lung tissues from untreated rats with steady-state levels of 5-7 adducts/109 nucleobases over a period of 70 weeks. This is the first study to report the detection of methyl DNA phosphate adducts in human lung tissues. The results provide new insights toward using these DNA adducts as potential biomarkers to study human exposure to environmental methylating carcinogens.
Assuntos
Cromatografia Líquida/métodos , Adutos de DNA/análise , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Fumar Tabaco/efeitos adversos , Animais , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Humanos , Pulmão/química , Pulmão/metabolismo , Neoplasias Pulmonares/química , Ratos , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodosRESUMO
Certain commensal and pathogenic bacteria produce colibactin, a small-molecule genotoxin that causes interstrand cross-links in host cell DNA. Although colibactin alkylates DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and ß-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line and incorporating two electrophilic cyclopropane warheads into the final natural product scaffold. This proposed transformation was supported by the detection of a colibactin-derived cross-linked DNA adduct. Overall, this work provides a biosynthetic explanation for colibactin's DNA cross-linking activity and paves the way for further study of its chemical structure and biological roles.
Assuntos
Amidoidrolases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeos/metabolismo , Policetídeos/metabolismo , Amidoidrolases/química , Domínio Catalítico , Ciclopropanos/química , Ciclopropanos/metabolismo , DNA Bacteriano/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Mutação , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
Acrolein, the simplest α,ß-unsaturated aldehyde, is present in relatively large quantities in cigarette smoke, and several studies have raised the possibility of it being a major etiological agent for smoking-related lung cancer. Acrolein reacts directly with DNA to form primarily Acr-dGuo adducts, which serve as important biomarkers for the assessment of exposure to acrolein and its potential role in smoking-related lung cancer. In this study, we developed an ultrasensitive and low-artifact method using liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry to quantitate Acr-dGuo adducts in normal lung tissue DNA obtained at surgery from lung cancer patients who never smoked and from those who continued smoking until surgery, as confirmed by urinary total cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. This provides a direct comparison of Acr-dGuo levels in human lung tissue as a result of cigarette smoking versus other etiological causes. There was no significant difference between the total Acr-dGuo levels in smokers (28.5 ± 14.9 adducts/109 nucleotides) and nonsmokers (25.0 ± 10.7 adducts/109 nucleotides), suggesting rapid removal of acrolein by glutathione conjugation and other detoxification mechanisms. Our results do not support the hypothesis that acrolein is a major etiological agent for cigarette smoking-related DNA damage.
Assuntos
Acroleína/química , Adutos de DNA/análise , DNA/química , Pulmão/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Acroleína/toxicidade , Cromatografia Líquida de Alta Pressão , Dano ao DNA/efeitos dos fármacos , Humanos , não Fumantes , FumantesRESUMO
Frequent exposure to chemicals in the environment, diet, and endogenous electrophiles leads to chemical modification of DNA and the formation of DNA adducts. Some DNA adducts can induce mutations during cell division and, when occurring in critical regions of the genome, can lead to the onset of disease, including cancer. The targeted analysis of DNA adducts over the past 30 years has revealed that the human genome contains many types of DNA damages. However, a long-standing limitation in conducting DNA adduct measurements has been the inability to screen for the total complement of DNA adducts derived from a wide range of chemicals in a single assay. With the advancement of high-resolution mass spectrometry (MS) instrumentation and new scanning technologies, nontargeted "omics" approaches employing data-dependent acquisition and data-independent acquisition methods have been established to simultaneously screen for multiple DNA adducts, a technique known as DNA adductomics. However, notable challenges in data processing must be overcome for DNA adductomics to become a mature technology. DNA adducts occur at low abundance in humans, and current softwares do not reliably detect them when using common MS data acquisition methods. In this perspective, we discuss contemporary computational tools developed for feature finding of MS data widely utilized in the disciplines of proteomics and metabolomics and highlight their limitations for conducting nontargeted DNA-adduct biomarker discovery. Improvements to existing MS data processing software and new algorithms for adduct detection are needed to develop DNA adductomics into a powerful tool for the nontargeted identification of potential cancer-causing agents.