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Talanta Jun 2024Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic...
Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.
Topics: Humans; Rats; Animals; DNA Adducts; Nucleosides; Chromatography, Liquid; Tandem Mass Spectrometry; DNA Damage; Liver; DNA; Organoids; Lansoprazole
PubMed: 38508126
DOI: 10.1016/j.talanta.2024.125902 -
Biomarkers : Biochemical Indicators of... May 2024Exocyclic DNA adducts have been shown to be potential biomarkers of cancer risk related to oxidative stress and exposure to aldehydes in smokers. In fact, aldehydes...
CONTEXT
Exocyclic DNA adducts have been shown to be potential biomarkers of cancer risk related to oxidative stress and exposure to aldehydes in smokers. In fact, aldehydes potentially arise from tobacco combustion directly and endogenously through lipid peroxidation.
OBJECTIVE
This study aims to investigate the relationship between a profile of nine aldehydes-induced DNA adducts and antioxidant activities, in order to evaluate new biomarkers of systemic exposure to aldehydes.
METHODS
Using our previously published UPLC-MS/MS method, adducts levels were quantified in the blood DNA of 34 active smokers. The levels of antioxidant vitamins (A, C and E), coenzyme Q10, β-carotene, superoxide dismutase (SOD) and autoantibodies against oxidized low-density lipoprotein were measured.
RESULTS
Adducts induced by tobacco smoking-related aldehydes were quantified at levels reflecting an oxidative production from lipid peroxidation. A significant correlation between SOD and crotonaldehyde-induced adducts ( = 0.0251) was also observed. β-Carotene was negatively correlated with the adducts of formaldehyde ( 0.0351) and acetaldehyde ( 0.0413). Vitamin C tended to inversely correlate with acetaldehyde-induced adducts ( = 0.0584).
CONCLUSION
These results are promising, and the study is now being conducted on a larger cohort with the aim of evaluating the impact of smoking cessation programs on the evolution of adducts profile and antioxidants activities.
Topics: Humans; DNA Adducts; Smokers; Biological Monitoring; Antioxidants; beta Carotene; Chromatography, Liquid; Tandem Mass Spectrometry; Aldehydes; Oxidative Stress; Biomarkers; Acetaldehyde; Superoxide Dismutase
PubMed: 38506499
DOI: 10.1080/1354750X.2024.2333361 -
Cell Biochemistry and Function Mar 2024The majority of adenocarcinoma lung cancer is found in nonsmokers. A history of tobacco use is more common in squamous cell carcinoma of the lung. The aim of this study...
The majority of adenocarcinoma lung cancer is found in nonsmokers. A history of tobacco use is more common in squamous cell carcinoma of the lung. The aim of this study is to identify the cisplatin (CDDP)-resistance that promotes lung squamous carcinoma cell growth through nicotine-mediated HDAC1/7nAchR/E2F/pRb cell cycle activation. Squamous cell carcinoma (NCI-H520 and NCI-H157) cells were examined after cisplatin and nicotine treatment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, cell migration assay, immunofluorescence staining, western blot analysis, and immunoprecipitation analysis. Consequently, CDDP is released from DNA and Rb phosphorylated pRb as a result of nicotine-induced cancer cell proliferation through 7nAchR, which then triggers the opening of the HDAC1 cell cycle. The cell cycle is stopped when CDDP adducts are present. Nicotine exerts cancer cytoprotective effects by allowing HDAC1 repair mechanisms to re-establish E2F promoting DNA stimulation cell cycle integrity in the cytosol and preventing potential CDDP and HDAC1 suppressed in the nuclear. Concentration expression of nicotine causes squamous carcinoma cell carcinogens to emerge from inflammation. COX2, NF-KB, and NOS2 increase as a result of nicotine-induced squamous carcinoma cell inflammation. Nicotine enhanced the cell growth-related proteins such as α7nAchR, EGFR, HDAC1, Cyclin D, Cyclin E, E2F, Rb, and pRb by western blot analysis. It also induced cancer cell inflammation and growth. As a result, we suggest that nicotine will increase the therapeutic resistance effects of CDDP. This has the potential to interact with nicotine through α7nAchR receptors and HDAC1/Cyclin D/E2F/pRb potentially resulting in CDDP therapy resistance, as well as cell cycle-induced cancer cell growth.
Topics: Humans; Cisplatin; Nicotine; alpha7 Nicotinic Acetylcholine Receptor; Cyclin D1; Cell Cycle; Carcinoma, Squamous Cell; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Lung Neoplasms; Lung; DNA; Inflammation; Cell Line, Tumor; Histone Deacetylase 1
PubMed: 38504444
DOI: 10.1002/cbf.3990 -
Chemical Research in Toxicology Apr 2024Aflatoxin B (AFB) is a potent human liver carcinogen produced by certain molds, particularly and , which contaminate peanuts, corn, rice, cottonseed, and ground and...
Aflatoxin B (AFB) is a potent human liver carcinogen produced by certain molds, particularly and , which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB undergoes bioactivation in the liver to produce AFB--8,9-epoxide, which forms the covalently bound cationic AFB-7-guanine (AFB7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B (AFB-FapyGua) adducts. The AFB formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB-7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB-FapyGua adducts formed in AFB-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS measurements can detect AFB-FapyGua at a quantification limit of 4.0 adducts per 10 bases when only 0.8 μg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.
Topics: Mice; Humans; Animals; DNA Adducts; Aflatoxin B1; Paraffin Embedding; DNA; Carcinogens; Mass Spectrometry; Guanine; Formaldehyde
PubMed: 38498000
DOI: 10.1021/acs.chemrestox.4c00005 -
Current Research in Toxicology 2024Colorectal cancer (CRC) is the third leading cause of cancer-related mortalities in the USA and around 52,550 people were expected to die from this disease by December...
Colorectal cancer (CRC) is the third leading cause of cancer-related mortalities in the USA and around 52,550 people were expected to die from this disease by December 2023. The objective of this study was to investigate the effect of diet type on benzo(a)pyrene [B(a)P]-induced colon cancer in an adult male rat model, the Polyposis In the Rat Colon (PIRC) kindred type. Groups of PIRC rats (n = 10) were fed with AIN-76A regular diet (RD) or Western diet (WD) and received 25, 50 and 100 µg B(a)P/kg body wt. via oral gavage for 60 days. Rats fed diets alone, but no B(a)P, served as controls. After exposure, rats were euthanized; colon and liver samples were analyzed for activation of drug metabolizing enzymes (DMEs) CYP1A1, CYP1B1, SULT and GST. Plasma and tissue samples were analyzed by reverse phase-HPLC for B(a)P metabolites. In addition to these studies, DNA isolated from colon and liver tissues was analyzed for B(a)P-induced DNA adducts by the P-postlabeling method using a thin-layer chromatography system. Western diet consumption resulted in a marked increase in DME expression and B(a)P metabolite concentrations in rats that were administered 100 µg/kg B(a)P + WD (p < 0.05) compared to other treatment groups. Our findings demonstrate that WD accelerates the development of colon tumors induced by B(a)P through enhanced biotransformation, and the products of this process (metabolites) were found to bind with DNA and form B(a)P-DNA adducts, which may have given rise to colon polyps characterized by gain in tumor number, sizes, and dysplasia.
PubMed: 38496007
DOI: 10.1016/j.crtox.2024.100162 -
Journal of Biological Inorganic... Mar 2024Organometallic η-arene ruthenium(II) complexes with 3-chloro-6-(1H-pyrazol-1-yl)pyridazine (Ru1, Ru2, and Ru5) and 3-chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine...
Organometallic η-arene ruthenium(II) complexes with 3-chloro-6-(1H-pyrazol-1-yl)pyridazine (Ru1, Ru2, and Ru5) and 3-chloro-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine (Ru3-4) N,N' heterocyclic and η-arene (cymene (Ru1-4) or toluene (Ru 5)) have been synthesized. The ruthenium(II) complexes have common "three-legged piano-stool" pseudo-octahedral structures known for half-sandwich complexes. Evolution of their UV-Visible absorption spectra in PBS buffer or DMSO over 24 h confirmed their good solvolysis stability. Titrations of the complexes with the calf thymus DNA (CT-DNA) were monitored using UV-Visible absorption and fluorescence spectroscopies. The complexes interact moderately with CT-DNA and their binding constants are in the order of 10 M. Competitive binding of the complexes to a DNA-Hoechst 33,258 depicted competitive displacement of Hoechst from DNA's minor grooves. These complexes bind to glutathione forming GSH-adducts through S coordination by replacement of a halide, with the iodo-analogues having higher binding constants than the chloro-complexes. Cyclic voltammograms of the complexes exhibited one electron-transfer quasi-reversible process. Trends in the molecular docking data of Ru1-5/DNA were similar to those for DNA binding constants. Of the five, only Ru1, Ru3 and Ru5 showed some activity (moderate) against the MCF-7 breast cancer cells with IC values in the range of 59.2-39.9 for which Ru5 was the most active. However, the more difficult-to-treat cell line, MDA-MB 231 cell was recalcitrant to the treatment by these complexes.
Topics: DNA; Humans; Ruthenium; Ligands; Coordination Complexes; Antineoplastic Agents; Glutathione; Cattle; Pyrazoles; Animals; Molecular Structure; Drug Screening Assays, Antitumor; Cell Proliferation; MCF-7 Cells; Cell Line, Tumor
PubMed: 38494554
DOI: 10.1007/s00775-024-02043-3 -
DNA Repair May 2024Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER,...
Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5'-deoxyribose phosphate (5'dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase β, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5'dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5'dRp residues at a rate comparable to that of DNA polymerase (pol) β, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.
Topics: Humans; DNA-(Apurinic or Apyrimidinic Site) Lyase; Lyases; Lysine; DNA Polymerase beta; DNA Repair; DNA Polymerase gamma; DNA, Mitochondrial; DNA-Binding Proteins; Transcription Factors; Mitochondrial Proteins; Phosphorus-Oxygen Lyases
PubMed: 38492429
DOI: 10.1016/j.dnarep.2024.103666 -
Food and Chemical Toxicology : An... May 2024Pyrrolizidine alkaloids (PA) are comprised of a family of hundreds of metabolites, produced by plants as a mechanism to protect against herbivory. Upon ingestion and...
Pyrrolizidine alkaloids (PA) are comprised of a family of hundreds of metabolites, produced by plants as a mechanism to protect against herbivory. Upon ingestion and metabolism, dehydropyrrolizidine alkaloids are formed, which are known to generate DNA adducts and subsequently double-strand DNA breaks. Within the liver, the most sensitive cell type to PA exposure is the sinusoidal endothelial cell, as evidenced by the generation of veno-occlusive disease in the human population. PAs are a common crop contaminant and have been regulated by some agencies, using the precautionary principle; each equally potent and genotoxic. Therefore, as a proof of principle we have established a human in vitro coculture model system, utilizing the metabolically active HepaRG hepatocyte and the SK-Hep-1 endothelial cell, to determine differential potencies of different PAs commonly found in crops and food products, notably cell death, targeting of endothelial cells, and genotoxicity comparing the micronucleus assay versus γH2AX assay. Our results demonstrate differential potencies of the PAs used, which encompass three esterification states (monoester, cyclic diester, and open-chain diester). The results suggest that a more nuanced approach to the regulation of PAs may be more appropriate in the regulatory decision-making process.
Topics: Humans; Pyrrolizidine Alkaloids; Endothelial Cells; Coculture Techniques; Hepatocytes; Liver
PubMed: 38490353
DOI: 10.1016/j.fct.2024.114584 -
Journal of Cancer Research and Clinical... Mar 2024Psoralen is a family of naturally occurring photoactive compounds found in plants that acquire potential cytotoxicity when activated by specific frequencies of... (Review)
Review
Psoralen is a family of naturally occurring photoactive compounds found in plants that acquire potential cytotoxicity when activated by specific frequencies of electromagnetic waves. Psoralens penetrate the phospholipid cellular membranes and insert themselves between the pyrimidines of deoxyribonucleic acid (DNA). Psoralens are initially biologically inert and acquire photoreactivity when exposed to certain classes of electromagnetic radiation, such as ultraviolet light. Once activated, psoralens form mono- and di-adducts with DNA, leading to marked cell apoptosis. This apoptotic effect is more pronounced in tumor cells due to their high rate of cell division. Moreover, photoactivated psoralen can inhibit tyrosine kinase signaling and influence the immunogenic properties of cells. Thus, the cytotoxicity of photoactivated psoralen holds promising clinical applications from its immunogenic properties to potential anti-cancer treatments. This narrative review aims to provide an overview of the current understanding and research on psoralen and to explore its potential future pharmacotherapeutic benefits in specific diseases.
Topics: Humans; Ficusin; Furocoumarins; Ultraviolet Rays; DNA
PubMed: 38489072
DOI: 10.1007/s00432-024-05648-y -
Chemistry (Weinheim An Der Bergstrasse,... May 2024Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites...
Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA-C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision-induced unfolding (CIU) studies via ion-mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein-RAPTA-C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N-terminal S-Ru-N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.
Topics: Ubiquitin; Myoglobin; Binding Sites; Cytochromes c; Muramidase; Protein Folding; Protein Binding; Ruthenium; Coordination Complexes
PubMed: 38472116
DOI: 10.1002/chem.202400268