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International Journal of Molecular... Dec 2022Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in...
Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, and , were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP--7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.
Topics: Humans; Activation, Metabolic; Benzo(a)pyrene; Cytochrome P-450 CYP1A1; DNA Adducts; Liver; Organoids
PubMed: 36614051
DOI: 10.3390/ijms24010606 -
Chemical Research in Toxicology Sep 2023Adductomics studies are used for the detection and characterization of various chemical modifications (adducts) of nucleic acids and proteins. The advancements in liquid...
Adductomics studies are used for the detection and characterization of various chemical modifications (adducts) of nucleic acids and proteins. The advancements in liquid chromatography coupled with high-resolution tandem mass spectrometry (HRMS/MS) have resulted in efficient methods for qualitative and quantitative adductomics. We developed an HRMS-based method for the simultaneous analysis of RNA and DNA adducts in a single run and demonstrated its application using Baltic amphipods, useful sentinels of environmental disturbances, as test organisms. The novelty of this method is screening for RNA and DNA adducts by a single injection on an Orbitrap HRMS instrument using full scan and data-independent acquisition. The MS raw files were processed with an open-source program, , to identify and distinguish RNA and DNA adducts based on the characteristic neutral loss of ribonucleosides and 2'-deoxyribonucleosides, respectively. In the amphipods, in addition to the nearly 150 putative DNA adducts characterized earlier, we detected 60 putative RNA adducts. For the structural identification of the detected RNA adducts, the MODOMICS database was used. The identified RNA adducts included simple mono- and dimethylation and other larger functional groups on different ribonucleosides and deaminated product inosine. However, 54 of these RNA adducts are not yet structurally identified, and further work on their characterization may uncover new layers of information related to the transcriptome and help understand their biological significance. Considering the susceptibility of nucleic acids to environmental factors, including pollutants, the developed multi-adductomics methodology with further advancement has the potential to provide biomarkers for diagnostics of pollution effects in biota.
Topics: DNA Adducts; RNA; DNA; Tandem Mass Spectrometry; Chromatography, Liquid
PubMed: 37566384
DOI: 10.1021/acs.chemrestox.3c00041 -
Toxicological Sciences : An Official... Jan 2022Vinyl acetate monomer (VAM) is heavily used to synthesize polymers. Previous studies have shown that inhaled VAM, being metabolized to acetaldehyde, may form DNA adducts...
Vinyl acetate monomer (VAM) is heavily used to synthesize polymers. Previous studies have shown that inhaled VAM, being metabolized to acetaldehyde, may form DNA adducts including N2-ethylidene-deoxyguanosine (N2-EtD-dG), which may subsequently cause mutations and contribute to its carcinogenesis. Currently, there is little knowledge on the molecular dosimetry between VAM exposure and DNA adducts under dosages relevant to human exposure. In this study, 0.02, 0.1, 1, 10, 50, 200, and 600 ppm VAM were exposed to rats by inhalation for 14 days (6 h/day). The use of [13C2]-VAM allows unambiguous differentiation and quantification of the exogenous and endogenous N2-EtD-dG by highly sensitive LC-MS/MS. Our data indicate that VAM-induced exogenous DNA adducts were formed in a non-linear manner. Exogenous DNA adducts were only detected in the nasal epithelium of rats exposed to 10, 50, 200, and 600 ppm VAM, whereas endogenous adducts were found in all nasal and other tissues analyzed. In addition, ratios of exogenous/endogenous DNA adducts were less than 1 with the dose up to 50 ppm, indicating that endogenous DNA adducts are predominant at low VAM concentrations. Moreover, differential dose-response in terms of exogenous DNA adduct formation were observed between nasal respiratory and olfactory epithelium. Furthermore, the lack of exogenous DNA adducts in distant tissues, including peripheral blood mononuclear cells, liver, brain, and bone marrow, indicates that VAM and/or its metabolite do not distribute systemically to cause DNA damage in distant tissues. Together, these results provided new molecular dosimetry to improve science-based cancer risk assessments of VAM.
Topics: Animals; Chromatography, Liquid; DNA Adducts; Leukocytes, Mononuclear; Rats; Tandem Mass Spectrometry; Vinyl Compounds
PubMed: 34904679
DOI: 10.1093/toxsci/kfab140 -
Trends in Molecular Medicine Feb 2018Alcohol use disorder (AUD) screening frequently involves questionnaires complemented by laboratory work to monitor alcohol use and/or evaluate AUD-associated... (Review)
Review
Alcohol use disorder (AUD) screening frequently involves questionnaires complemented by laboratory work to monitor alcohol use and/or evaluate AUD-associated complications. Here we suggest that measuring aldehyde-induced DNA and protein adducts produced during alcohol metabolism may lead to earlier detection of AUD and AUD-associated complications compared with existing biomarkers. Use of aldehyde-induced adducts to monitor AUD may also be important when considering that approximately 540 million people bear a genetic variant of aldehyde dehydrogenase 2 (ALDH2) predisposing this population to aldehyde-induced toxicity with alcohol use. We posit that measuring aldehyde-induced adducts may provide a means to improve precision medicine approaches, taking into account lifestyle choices and genetics to evaluate AUD and AUD-associated complications.
Topics: Alcoholism; Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Biomarkers; DNA Adducts; Humans; Mutation
PubMed: 29422263
DOI: 10.1016/j.molmed.2017.12.003 -
Chemical Research in Toxicology Oct 2022Bacteria harboring glycerol/diol dehydratase (GDH) encoded by the genes CDE metabolize glycerol and release acrolein during growth. Acrolein has antimicrobial activity,...
Bacteria harboring glycerol/diol dehydratase (GDH) encoded by the genes CDE metabolize glycerol and release acrolein during growth. Acrolein has antimicrobial activity, and exposure of human cells to acrolein gives rise to toxic and mutagenic responses. These biological responses are related to acrolein's high reactivity as a chemical electrophile that can covalently bind to cellular nucleophiles including DNA and proteins. Various food microbes and gut commensals transform glycerol to acrolein, but there is no direct evidence available for bacterial glycerol metabolism giving rise to DNA adducts. Moreover, it is unknown whether pathogens, such as Typhymurium, catalyze this transformation. We assessed, therefore, acrolein formation by four GDH-competent strains of Typhymurium grown under either aerobic or anaerobic conditions in the presence of 50 mM glycerol. On the basis of analytical derivatization with a heterocyclic amine, all wild-type strains were observed to produce acrolein, but to different extents, and acrolein production was not detected in fermentations of a C-deficient mutant strain. Furthermore, we found that, in the presence of calf thymus DNA, acrolein-DNA adducts were formed as a result of bacterial glycerol metabolism by two strains of , but not a CDE mutant strain. The quantification of the resulting adducts with increasing levels of glycerol up to 600 mM led to the production of up to 1.5 mM acrolein and 3600 acrolein-DNA adducts per 10 nucleosides in a model system. These results suggest that GDH-competent food microbes, gut commensals, and pathogens alike have the capacity to produce acrolein from glycerol. Further, the acrolein production can lead to DNA adduct formation, but requires high glycerol concentrations that are not available in the human gut.
Topics: Acrolein; Amines; Anti-Infective Agents; Bacteria; DNA; DNA Adducts; Glycerol; Humans; Propanediol Dehydratase
PubMed: 36116084
DOI: 10.1021/acs.chemrestox.2c00137 -
Mutation Research. Reviews in Mutation... 2016From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation... (Review)
Review
From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations.
Topics: Alkylating Agents; Alkylation; Apoptosis; DNA Adducts; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Humans; Mutagenicity Tests
PubMed: 27036068
DOI: 10.1016/j.mrrev.2015.11.001 -
Analytical Chemistry Mar 2022Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause...
Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life-threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with three drug/protein combinations and apply our label-free workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing label-free tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools.
Topics: Algorithms; DNA Adducts; Mass Spectrometry; Proteins; Proteomics; Software
PubMed: 35184559
DOI: 10.1021/acs.analchem.1c04101 -
DNA Repair Jan 2020Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid...
Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxycytosine. Excision kinetics on DNA containing a site-specific aflatoxin B-FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.
Topics: Adenosine Deaminase; Amino Acid Substitution; Catalytic Domain; DNA Adducts; DNA Glycosylases; Gas Chromatography-Mass Spectrometry; Gene Editing; Humans; Models, Molecular; Molecular Weight; Protein Conformation; RNA-Binding Proteins; Substrate Specificity
PubMed: 31733589
DOI: 10.1016/j.dnarep.2019.102741 -
Aging Jul 2023Chronic inflammation and lipid peroxidation (LPO) are associated with the pathogenesis of hepatocellular carcinoma (HCC), and γ-hydroxy-1, N-propanodeoxyguanosine...
PURPOSE
Chronic inflammation and lipid peroxidation (LPO) are associated with the pathogenesis of hepatocellular carcinoma (HCC), and γ-hydroxy-1, N-propanodeoxyguanosine (γ-OHPdG) is a promutagenic DNA adduct derived from LPO. This study aimed to examine the relationship between γ-OHPdG and the progression of liver carcinogenesis.
METHODS
Primary HCC specimens were obtained from 228 patients and cirrhosis specimens from 46 patients. The patients were followed up with after surgery via outpatient visits and telephone calls. The levels of γ-OHPdG were determined by immunohistochemical analysis in the carcinomatous tissues together with adjacent and cirrhosis tissues.
RESULTS
γ-OHPdG levels in the cancerous tissues were significantly higher compared to adjacent tissues (P < 0.001) and also higher than the ones from the tissues of cirrhosis patients. Along with tumor size, histological grade, MVI grade, T stage, the percentage of ki67-positive cells and HCC progression, γ-OHPdG levels in cancerous tissues showed a gradually increasing trend. Moreover, prognostic analysis showed that higher γ-OHPdG levels in cancerous tissues were strongly correlated with lower overall survival (P < 0.001), lower intrahepatic recurrence-free survival (P < 0.001) and lower distant metastasis-free survival (P < 0.05). There was a trend, although not statistically significant, of increased levels of γ-OHPdG in cirrhosis cases that advanced to HCC, whereas γ-OHPdG levels reversely correlated with the period of time observed for cirrhosis advanced to HCC.
CONCLUSIONS
These results suggest that γ-OHPdG is a prognostic biomarker for predicting outcomes in HCC, and may serve as a prospective indicator for predicting HCC in cirrhosis patients.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; DNA Adducts; Prognosis; Lipid Peroxidation; Prospective Studies; Liver Cirrhosis; Biomarkers; Biomarkers, Tumor
PubMed: 37517089
DOI: 10.18632/aging.204910 -
Nature Jun 2024DNA base damage is a major source of oncogenic mutations. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of...
DNA base damage is a major source of oncogenic mutations. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.
Topics: DNA Damage; DNA Repair; Mutagenesis; DNA Replication; Mutation; Humans; Animals; DNA Adducts; Ultraviolet Rays; DNA; Alkylation; DNA-Directed DNA Polymerase
PubMed: 38867042
DOI: 10.1038/s41586-024-07490-1