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Scientific Reports Aug 20236-Thioguanine is an immunosuppressive drug, an analogue of guanine, applied to treat acute leukemia and inflammatory bowel disease. Excessive use of 6-thioguanine during...
6-Thioguanine is an immunosuppressive drug, an analogue of guanine, applied to treat acute leukemia and inflammatory bowel disease. Excessive use of 6-thioguanine during clinical treatment may cause side effects. Moreover, providing a dose too low will be ineffective. Therefore, there is a critical need for a rapid, selective and routine approach to quantifying 6-thioguanine in body fluids to support a clinical application. A fully validated HPLC method has been developed to determine 6-thioguanine in whole blood samples using 5-bromouracil as an internal standard. 6-Thioguanine nucleotides were released from erythrocytes by perchloric acid, and then hydrolysed at 100 °C to the parent thiopurine, 6-thioguanine. The following validation parameters of the method were determined: specificity/selectivity, linearity range (479-17,118 ng/mL, R > 0.992), limits of detection (150 ng/mL) and quantification (479 ng/mL), accuracy (- 5.6 < Bias < 14.7), repeatability (CV 1.30-3.24%), intermediate precision (CV 4.19-5.78%), extraction recovery (79.1-103.6%) and carryover. Furthermore, the stability of the drug in whole blood samples under various storage conditions was investigated. The suggested method is suitable for determining 6-thioguanine in whole blood erythrocyte samples for drug level monitoring, thus correct dosing.
Topics: Thioguanine; Chromatography, High Pressure Liquid; Erythrocytes; Body Fluids; Bromouracil
PubMed: 37644112
DOI: 10.1038/s41598-023-41426-5 -
International Journal of Molecular... May 2023When modified uridine derivatives are incorporated into DNA, radical species may form that cause DNA damage. This category of molecules has been proposed as...
When modified uridine derivatives are incorporated into DNA, radical species may form that cause DNA damage. This category of molecules has been proposed as radiosensitizers and is currently being researched. Here, we study electron attachment to 5-bromo-4-thiouracil (BrSU), a uracil derivative, and 5-bromo-4-thio-2'-deoxyuridine (BrSdU), with an attached deoxyribose moiety via the N-glycosidic (N1-C) bond. Quadrupole mass spectrometry was used to detect the anionic products of dissociative electron attachment (DEA), and the experimental results were supported by quantum chemical calculations performed at the M062X/aug-cc-pVTZ level of theory. Experimentally, we found that BrSU predominantly captures low-energy electrons with kinetic energies near 0 eV, though the abundance of bromine anions was rather low compared to a similar experiment with bromouracil. We suggest that, for this reaction channel, proton-transfer reactions in the transient negative ions limit the release of bromine anions.
Topics: Electrons; Deoxyribose; Bromine; Anions; Bromodeoxyuridine
PubMed: 37240053
DOI: 10.3390/ijms24108706 -
Heliyon Mar 2023A rapid, specific and accurate high-performance liquid chromatography with tunable ultraviolet detection method was developed to simultaneously determine azathioprine...
A rapid, specific and accurate high-performance liquid chromatography with tunable ultraviolet detection method was developed to simultaneously determine azathioprine metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine riboside (6-MMPr) in human red blood cells. Erythrocyte lysate sample was precipitated by perchloric acid under the protection of dithiothreitol, with 6-TGN and 6-MMPr being acid hydrolyzed to produce 6-thioguanine (6-TG) and 6-methymercaptopurine (6-MMP). A Waters Cortecs C column (2.1 × 150 mm, 2.7 μm) was used for chromatographic separation with a water (containing 0.01 mol/L ammonium acetate and 0.2% acetic acid)/methanol linear gradient at a flow rate of 0.45 mL/min in a 5.5 min. UV detection wavelengths were 340 nm for 6-TG, 303 nm for 6-MMP and the IS (5-bromouracil). The calibration curves fitted a least squares model (weighed 1/ ) from 0.15 to 15 μmol/L for 6-TG ( = 0.9999) and from 1 to 100 μmol/L for 6-MMP ( = 0.9998). This method was validated according to the FDA bioanalytical method validation guidance and ICH M10 bioanalytical method validation and study sample analysis guidance for industry, and successfully utilized in ten IBD patients receiving azathioprine therapy.
PubMed: 36895397
DOI: 10.1016/j.heliyon.2023.e13870 -
Chembiochem : a European Journal of... Mar 2022In this study, we investigated the photoreaction of U in a pyrene-labeled DNA duplex, RNA duplex, and DNA/RNA hybrids. We found that the photoreactivity of U changed...
In this study, we investigated the photoreaction of U in a pyrene-labeled DNA duplex, RNA duplex, and DNA/RNA hybrids. We found that the photoreactivity of U changed dramatically from hydrogen abstraction to cross-linking by changing the conformation of the duplex from the B-form to the A-form. Among three A-form structures, the largest amount of cross-linked products was observed when U was incorporated into the RNA strand and the pyrene was conjugated to the 5' end of the DNA. These results indicate that the contact manner of pyrene was different between A- and B-form duplexes. This is a rare example of the use of the reactivity of bromouracil to analyze the contact between a small molecule with a weak binding affinity and a nucleic acid.
Topics: Circular Dichroism; DNA; Nucleic Acid Conformation; Pyrenes; RNA
PubMed: 35080796
DOI: 10.1002/cbic.202100626 -
Photochemistry and Photobiology May 20225-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage.... (Review)
Review
5-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage. In DNA strands, the uracil radical generated from 5-halouracil causes DNA damage via a hydrogen-abstraction reaction. We analyzed the photoreaction of 5-halouracil in various DNA structures and revealed that the reaction is DNA structure-dependent. In this review, we summarize the results of the analysis of the reactivity of 5-halouracil in various DNA local structures. Among the 5-halouracil molecules, 5-bromouracil has been used as a probe in the analysis of photoinduced electron transfer through DNA. The analysis of groove-binder/DNA and protein/DNA complexes using a 5-bromouracil-based electron transfer system is also described.
Topics: Bromouracil; DNA; Thymine; Uracil
PubMed: 34543451
DOI: 10.1111/php.13521 -
International Journal of Environmental... Mar 2021Chlorpyrifos, Bromacil and Terbuthylazine are commonly used as insecticides and herbicides to control weeds and prevent non-desirable growth of algae, fungi and bacteria...
Chlorpyrifos, Bromacil and Terbuthylazine are commonly used as insecticides and herbicides to control weeds and prevent non-desirable growth of algae, fungi and bacteria in many agricultural applications. Despite their highly negative effects on human health, environmental modeling of these pesticides in the vadose zone until they reach groundwater is still not being conducted on a regular basis. This work shows results obtained by version 5.08 of the Pesticide Root Zone Model (PRZM5) numerical model to simulate the fate and transport of Chlorpyrifos, Bromacil and Terbuthylazine between 2006 and 2018 inside the Buñol-Cheste aquifer in Spain. The model uses a whole set of parameters to solve a modified version of the mass transport equation considering the combined effect of advection, dispersion and reactive transport processes. The simulation process was designed for a set of twelve scenarios considering four application doses for each pesticide. Results show that the maximum concentration value for every scenario exceeds the current Spanish Maximum Concentration Limit (0.1 μg/L). Numerical simulations were able to reproduce concentration observations over time despite the limited amount of available data.
Topics: Bromouracil; Chlorpyrifos; Groundwater; Humans; Spain; Triazines; Water Pollutants, Chemical
PubMed: 33800654
DOI: 10.3390/ijerph18073511 -
PloS One 2021Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug...
Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.
Topics: Amyotrophic Lateral Sclerosis; Base Sequence; Bromouracil; Cloning, Molecular; Drug Evaluation, Preclinical; Escherichia coli; Humans; Hydrogen Bonding; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Protein Folding; Proton Magnetic Resonance Spectroscopy; Superoxide Dismutase-1; Trifluridine; Tryptophan; Uridine
PubMed: 33635895
DOI: 10.1371/journal.pone.0247684 -
Genome Research Oct 2020Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this,...
Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this, simultaneous measurements of RNA synthesis and degradation are required. Here, we report the development of "Dyrec-seq," which uses 4-thiouridine and 5-bromouridine to simultaneously quantify RNA synthesis and degradation rates. Dyrec-seq enabled the quantification of RNA synthesis and degradation rates of 4702 genes in HeLa cells. Functional enrichment analysis showed that the RNA synthesis and degradation rates of genes are actually determined by the genes' biological functions. A comparison of theoretical and experimental analyses revealed that the amount of RNA is determined by the ratio of RNA synthesis to degradation rates, whereas the rapidity of responses to external stimuli is determined only by the degradation rate. This study emphasizes that not only RNA synthesis but also RNA degradation is important in shaping gene expression patterns.
Topics: Bromouracil; HeLa Cells; Humans; RNA; Sequence Analysis, RNA; Thiouridine; Uridine
PubMed: 32843354
DOI: 10.1101/gr.264408.120 -
Molecules (Basel, Switzerland) Nov 2019Homonucleoside analogues - and - having a (5-methoxycarbonyl)isoxazolidine framework were synthesized via the 1,3-dipolar cycloaddition of nucleobase-derived nitrones...
Homonucleoside analogues - and - having a (5-methoxycarbonyl)isoxazolidine framework were synthesized via the 1,3-dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate. Hydrogenolysis of the isoxazolidines containing thymine, dihydrouracil, theophylline and adenine moieties efficiently led to the formation of the respective γ-lactam analogues. γ-Lactam analogues having 5-bromouracil and 5-chlorouracil fragments were synthesized by treatment of uracil-containing γ-lactams with NBS and NCS. Isoxazolidine and γ-lactam analogues of homonucleosides obtained herein were evaluated for activity against a broad range of DNA and RNA viruses. None of the compounds that were tested exhibited antiviral or cytotoxic activity at concentrations up to 100 µM. The cytostatic activities of all compounds toward nine cancerous cell lines was tested. γ-Lactams - (Cl-Ura) and - (Theo) appeared the most active toward pancreatic adenocarcinoma cells (Capan-1), showing IC values 21.5 and 18.2 µM, respectively. Isoxazolidine - (Cl-Ura) inhibited the proliferation of colorectal carcinoma (HCT-116).
Topics: Antineoplastic Agents; Isoxazoles; Lactams; Molecular Structure; Nucleosides; Spectrum Analysis
PubMed: 31698778
DOI: 10.3390/molecules24224014 -
International Journal of Molecular... Jul 2019Low-energy electrons (LEEs) of energies ≤30 eV are generated in large quantities by ionizing radiation. These electrons can damage DNA; particularly, they can induce... (Review)
Review
Low-energy electrons (LEEs) of energies ≤30 eV are generated in large quantities by ionizing radiation. These electrons can damage DNA; particularly, they can induce the more detrimental clustered lesions in cells. This type of lesions, which are responsible for a large portion of the genotoxic stress generated by ionizing radiation, is described in the Introduction. The reactions initiated by the collisions of 0.5-30 eV electrons with oligonucleotides, duplex DNA, and DNA bound to chemotherapeutic platinum drugs are explained and reviewed in the subsequent sections. The experimental methods of LEE irradiation and DNA damage analysis are described with an emphasis on the detection of cluster lesions, which are considerably enhanced in DNA-Pt-drug complexes. Based on the energy dependence of damage yields and cross-sections, a mechanism responsible for the clustered lesions can be attributed to the capture of a single electron by the electron affinity of an excited state of a base, leading to the formation of transient anions at 6 and 10 eV. The initial capture is followed by electronic excitation of the base and dissociative attachment-at other DNA sites-of the electron reemitted from the temporary base anion. The mechanism is expected to be universal in the cellular environment and plays an important role in the formation of clustered lesions.
Topics: Antineoplastic Agents; Bromouracil; Carboplatin; Cisplatin; DNA; DNA Breaks, Double-Stranded; Electrons; Eukaryotic Cells; Humans; Oligonucleotides; Oxaliplatin; Plasmids; Radiation, Ionizing; Radiation-Sensitizing Agents
PubMed: 31370253
DOI: 10.3390/ijms20153749